CD37-binding molecules and immunoconjugates thereof

ABSTRACT

Methods of using CD37 agents, including, but not limited to, antibodies and immunoconjugates, that bind to CD37 to deplete B-cells (e.g., non-cancerous B-cells) and methods of treating autoimmune and inflammatory diseases are further provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/436,528, filed Mar. 30, 2012, which claims the priority benefit ofU.S. Provisional Application No. 61/470,863, filed Apr. 1, 2011, each ofwhich is herein incorporated by reference in its entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name:2921_0170001_SEQIDListing.ascii.txt, Size: 233,101 bytes, and Date ofCreation: Jun. 12, 2012) is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The field of the invention generally relates to antibodies,antigen-binding fragments thereof, polypeptides, and immunoconjugatesthat bind to CD37, as well as to methods of using such CD37-bindingmolecules for the treatment of diseases, such as autoimmune diseases andinflammatory diseases.

BACKGROUND OF THE INVENTION

Leukocyte antigen CD37 (“CD37”), also known as GP52-40, tetraspanin-26,or TSPAN26, is a transmembrane protein of the tetraspanin superfamily(Maecker et al., 1997 FASEB J. 11:428-442). It is a heavily glycosylatedprotein with four transmembrane domains that is expressed on B cellsduring the pre-B to peripheral mature B-cell stages, but is reportedlyabsent on terminal differentiation to plasma cells. (Link et al., 1987,J Pathol. 152:12-21). The CD37 antigen is only weakly expressed onT-cells, myeloid cells, and granulocytes (Schwartz-Albiez et al. 1988,J. Immunol., 140(3)905-914). However, CD37 is also expressed onmalignant B-cells such as those founding non-Hodgkin's lymphoma (NHL)and chronic lymphoid leukemia (CLL) (Moore et al. 1986, J Immunol.137(9):3013-8).

While the exact physiological role of CD37 is unclear, studies inCD37-deficient mice suggest an immunoregulatory function. Although micedeficient in CD37 expression have normal development (Knobeloch et al.2000, Mol Cell Biol., 20(15):5363-9), in the C57/B16 background, CD37−/−T cells are hyper-proliferative (van Spriel et al., J Immunol. 172, 2953(2004)), CD37−/− dendritic cells (DC) exhibit an increased antigenpresentation (Sheng et al., Eur J Immunol. 39, 50 (2009)), and CD37−/−macrophages show increased dectin-1-induced IL-6 production(Meyer-Wentrup et al., J Immunol. 178, 154 (2007)). CD37-deficientC57/B16 mice also contain significantly higher level of IgA than thewild-type mice (van Spriel et al., PLoS Pathol. 5, e1000338 (2009) andRops et al., Am J Pathol. 176, 2188 (2010)). All of these resultssuggest a general regulatory role of CD37 in the immune system.Interestingly, crosslinking of CD37 antigen by antibody on human T cellsinhibits T cell proliferation induced by CD3 stimulation (van Spriel etal., J Immunol. 172, 2953 (2004)).

Antibodies are emerging as a promising method to treat human diseasesincluding autoimmune diseases. Currently, an anti-CD20 antibody calledrituximab has been approved for rheumatoid arthritis (RA) treatment(Edwards J C et al. 2006, Nat Rev Immunol. 6: 119). Rituximab is used inthe United States in combination with methotrexate (MTX) to reduce signsand symptoms in adult patients with moderately- to severely-active RAwho have had an inadequate response to at least one TNF antagonist. Manystudies address the use of rituximab in a variety of non-malignantautoimmune or inflammatory disorders, including RA, in which B-cells andautoantibodies appear to play a role in disease pathophysiology. Edwardset al., Biochem Soc. Trans. 30:824-828 (2002). Targeting of CD20 usinganti-CD20 antibody has been reported to potentially relieve signs andsymptoms of a number of autoimmune or inflammatory diseases including,for example, RA (Leandro et al., Ann. Rheum. Dis. 61:883-888 (2002);Edwards et al., Arthritis Rheum., 46 (Suppl. 9): S46 (2002); Stahl etal., Ann. Rheum. Dis., 62 (Suppl. 1): OP004 (2003); Emery et al.,Arthritis Rheum. 48(9): S439 (2003)), lupus (Eisenberg, Arthritis. Res.Ther. 5:157-159 (2003); Leandro et al. Arthritis Rheum. 46: 2673-2677(2002); Gorman et al., Lupus, 13: 312-316 (2004)), immunethrombocytopenic purpura (D'Arena et al., Leuk. Lymphoma 44:561-562(2003); Stasi et al., Blood, 98: 952-957 (2001); Saleh et al., Semin.Oncol., 27 (Supp 12):99-103 (2000); Zaja et al., Haematologica,87:189-195 (2002); Ratanatharathorn et al., Ann. Int. Med., 133:275-279(2000)), pure red cell aplasia (Auner et al., Br. J. Haematol.,116:725-728 (2002)); autoimmune anemia (Zaja et al., supra (erratumappears in Haematologica 87:336 (2002)), cold agglutinin disease (Layioset al., Leukemia, 15:187-8 (2001); Berentsen et al., Blood, 103:2925-2928 (2004); Berentsen et al., Br. J. Haematol., 115:79-83 (2001);Bauduer, Br. J. Haematol., 112:1083-1090 (2001); Zaja et al., Br. J.Haematol., 115:232-233 (2001)), type B syndrome of severe insulinresistance (Coll et al., N. Engl. J. Med., 350:310-311 (2004), mixedcryoglobulinermia (DeVita et al., Arthritis Rheum. 46 Suppl. 9:S206/S469(2002)), myasthenia gravis (Zaja et al., Neurology, 55:1062-1063 (2000);Wylam et al., J. Pediatr., 143:674-677 (2003)), Wegener's granulomatosis(Specks et al., Arthritis & Rheumatism 44:2836-2840 (2001)), microscopicpolyangiitis (MPA), refractory pemphigus vulgaris (Dupuy et al., ArchDermatol., 140:91-96 (2004)), dermatomyositis (Levine, Arthritis Rheum.,46 (Suppl. 9):51299 (2002)), Sjogren's syndrome (Somer et al., Arthritis& Rheumatism, 49:394-398 (2003)), active type-II mixed cryoglobulinemia(Zaja et al., Blood, 101:3827-3834 (2003)), pemphigus vulgaris (Dupay etal., Arch. Dermatol., 140:91-95 (2004)), autoimmune neuropathy (Pestronket al., J. Neurol. Neurosurg. Psychiatry 74:485-489 (2003)),paraneoplastic opsoclonus-myoclonus syndrome (Pranzatelli et al.Neurology 60 (Suppl. 1) PO5.128:A395 (2003)), and relapsing-remittingmultiple sclerosis (RRMS). Cross et al. (abstract) “Preliminary Resultsfrom a Phase II Trial of Rituximab in MS” Eighth Annual Meeting of theAmericas Committees for Research and Treatment in Multiple Sclerosis,20-21 (2003).

In animal models, B-cell depletion using antibodies against B-cellantigens such as CD20 has been shown to inhibit or ameliorate severalautoimmune diseases including systemic lupus erythematosus (SLE),experimental autoimmune encephalomyelitis (EAE; mouse model of multiplesclerosis), type-1 diabetes (T1D) and rheumatoid arthritis (RA).Rituximab has been shown to deplete both malignant and normal B cells invivo in animal models as well as patients (Maloney D G et al, Blood.1994; 84(8):2457-66; Reff M E, et al. Blood. 1994; 83(2):435-45;Schroder C, et al. Transpl Immunol. 2003; 12(1):19-28). It can alsodeplete normal B-cells from human peripheral blood mononuclear cells(PBMCs) in in vitro experiments (Vugmeyster Y, et al, Cytometry A. 2003;52(2):101-9; Vugmeyster Y and Howell K. Int Immunopharmacol. 2004;4(8):1117-24).

Campath-1H (alumtuzumab), an anti-CD52 chimeric IgG1, binds to the CD52antigen, which is highly expressed on all lymphocytes (Ginaldi L, et al,Leuk Res. 1998 February; 22(2):185-91; Hale G, et al, Tissue Antigens.1990 March; 35(3):118-27). It is used in patients to deplete malignantlymphocytes and is approved for treating chronic lymphocytic leukemia.It has also shown efficacy in treating multiple sclerosis and iscurrently in Phase III clinical testing (N Engl J Med 2008;359:1786-1801; ClinicalTrials.gov NCT00530348 & NCT00548405). It hasbeen shown to deplete normal lymphocytes in vitro as well (Hale G, etal. Blood. 1983 October; 62(4):873-82; Waldmann H and Hale G PhilosTrans R Soc Lond B Biol Sci. 2005 Sep. 29; 360(1461):1707-11).

CD37-binding agents are also being tested as potential therapeutics forB-cell malignancies. Emergent Biosolutions (formerly TrubionPharmaceuticals) developed the CD37-binding agents SMIP-016 and TRU-016(Zhao et al., 2007, Blood, 110:2569-2577). SMIP-016 is a single chainpolypeptide that includes variable regions from a hybridoma andengineered human constant regions. TRU-016 is a humanized version of theanti-CD37 SMIP protein. See e.g. U.S. Published Application No.2007/0059306. TRU-016 is being tested clinically for the treatment ofchronic lymphocytic leukemia (CLL). Boehringer Ingelheim has alsodisclosed a CD37 binding agent in International Published ApplicationNo. WO 2009/019312. However, no CDC activity has been described for anyof these binding agents and no in vitro pro-apoptotic activity has beendescribed in the absence of cross-linking agents.

Radio-immunotherapy (RIT) has been attempted using a radio-labeledanti-CD37 antibody MB-1 in two separate trials. Therapeutic doses of¹³¹I-MB-1 were administered to six relapsed NHL patients (Press et al.1989 J Clin Oncol. 7(8):1027-38; Press at el. 1993, N Engl J Med.329(17):1219-24). All six patients achieved a complete remission (CR)with a duration of four to thirty-one months. In another trial,¹³¹I-MB-1 was administered to ten relapsed NHL patients (Kaminski et al.1992 J Clin Oncol. 10(11):1696-711). A total of four patients had aresponse ranging in duration from two to six months, although only oneCR was reported. However, not all patients could be treated due to anunfavorable biodistribution of the radio-label which raised concern forradiation exposure of vital non-target organs. Indeed, RIT relatedtoxicities were observed in these trials including severemyelosupression and cardiopulmonary toxicity. While these clinical datasuggest that anti-CD37 radio-immunoconjugates may be effective, thesetherapies are cumbersome to administer, and at relapse post-RIT patientscannot be retreated with RIT due to the risks associated with high dosesof radiation.

To overcome the limitations of RIT, antibody-cytotoxic agent conjugates(ACC), also called antibody-drug conjugates (ADC), have been developed.These are immunoconjugates that include a cytotoxic agent covalentlylinked to an antibody through a chemical linker which can allow forspecific delivery of cytotoxic drugs to cells expressing a proteinrecognized by the antibody. However, proteins that are poorlyinternalized are not considered to be favorable targets for suchtherapeutics. CD37 is structurally similar to CD20 as both antigenscontain four transmembrane domains, although CD20 is not part of thetetraspanin family (Tedder et al. 1989, J. Immun. 142: 2560-2568).Antibodies against several B-cell antigens including CD37 and CD20 havebeen studied for their ability to undergo endocytosis and degradation(Press et al. 1989, Cancer Res. 49(17):4906-12, and Press et al. 1994,Blood. 83(5):1390-7). The anti-CD37 antibody MB-1 was retained on thecell surface and internalized slowly in Daudi lymphoma cells in vitro.The MB-1 antibody also had a low rate of endocytosis and intracellularmetabolism in NHL patient cells in vitro. Similar results were obtainedwith the anti-CD20 antibody 1F5, which was also retained mainly on thelymphoma cell surface and internalized poorly. ADCs of CD20 antibodieshave been studied previously but have not demonstrated significantlystrong potency, especially when non-disulfide or acid stable linkers areused (see for example Polson et al., 2009, Cancer Res.,69(6):2358-2364). In light of these observations, CD37 has not beenconsidered a favorable target for antibody-drug conjugates.

While their role in cancer treatment has been studied, the potentialeffect of CD37-directed therapies such as antibodies, antibodyderivatives or radio-immunoconjugates on cells involved in autoimmunediseases, inflammatory diseases or other disorders of the immune systemis not well understood. Furthermore, none of the compounds describedabove have been demonstrated to induce depletion of target cellsinvolved in manifestation or progression of these types of diseases.

Therefore, there exists a need for CD37 binding agents includingantibodies, antigen-binding fragments thereof, and antibody-drugconjugates (immunoconjugates) as a means to treat autoimmune diseases,inflammatory diseases, or other disorders of the immune system. Thepresent invention addresses that need.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present disclosure provides a method for depletingB-cells or treating a disease associated with aberrant B-cell activity,comprising administering to a patient an effective amount of a humanizedCD37 targeting antibody or immunoconjugate provided herein. In someembodiments, the B-cells are non-cancerous B-cells. In some embodiments,the B-cells do not overexpress CD37.

In certain embodiments, the disease associated with aberrant B-cellactivity is a disease associated with B-cell autoantibody production,and/or a disease associated with inappropriate T-cell stimulation inconnection with a B-cell pathway.

In certain embodiments, the disease characterized by autoantibodyproduction is rheumatoid arthritis, multiple sclerosis, type I diabetesmellitus, idiopathic inflammatory myopathy, systemic lupus erythematosus(SLE), myasthenia gravis, Grave's disease, dermatomyositis,polymyositis, or other autoimmune diseases.

In certain embodiments, the present disclosure provides a method fordepleting a B-cell comprising contacting a B-cell (e.g., in a populationof cells comprising a non-cancerous B-cell) with an antibody or antigenbinding fragment thereof that specifically binds to CD37, wherein theantibody or fragment thereof is capable of inducing apoptosis in vitroin the absence of a cross-linking agent. In certain embodiments, thepresent disclosure provides a method for treating a patient having anautoimmune or inflammatory disease comprising administering to thepatient a therapeutically effective amount of an antibody orantigen-binding fragment thereof that specifically binds to CD37,wherein the antibody or fragment thereof is capable of inducingapoptosis in vitro in the absence of a cross-linking agent. In someembodiments, the antibody or antigen-binding fragment thereof is alsocapable of inducing complement dependent cytotoxicity (CDC). In someembodiments, the antibody or antigen-binding fragment thereof is alsocapable of inducing antibody dependent cell mediated cytotoxicity(ADCC). In some embodiments, the antibody or antigen-binding fragmentthereof has a long serum half-life.

In certain embodiments, the present disclosure provides a method fordepleting a B-cell comprising contacting a B-cell (e.g., in a populationof cells comprising a non-cancerous B-cell) with an antibody or antigenbinding fragment thereof that specifically binds to the same CD37epitope as an antibody selected from the group consisting of: (a) anantibody comprising the polypeptide of SEQ ID NO:55 and the polypeptideof SEQ ID NO:72; (b) an antibody comprising the polypeptide of SEQ IDNO:56 and the polypeptide of SEQ ID NO:73; (c) an antibody comprisingthe polypeptide of SEQ ID NO:57 and the polypeptide of SEQ ID NO:74; (d)an antibody comprising the polypeptide of SEQ ID NO:58 and thepolypeptide of SEQ ID NO:74; (e) an antibody comprising the polypeptideof SEQ ID NO:59 and the polypeptide of SEQ ID NO:75; (f) an antibodycomprising the polypeptide of SEQ ID NO:60 and the polypeptide of SEQ IDNO:76; (g) an antibody comprising the polypeptide of SEQ ID NO:61 andthe polypeptide of SEQ ID NO:77; (h) an antibody comprising thepolypeptide of SEQ ID NO:62 and the polypeptide of SEQ ID NO:78; (i) anantibody comprising the polypeptide of SEQ ID NO:63 and the polypeptideof SEQ ID NO:79; (j) an antibody comprising the polypeptide of SEQ IDNO:64 and the polypeptide of SEQ ID NO:80; (k) an antibody comprisingthe polypeptide of SEQ ID NO:65 and the polypeptide of SEQ ID NO:81; (l)an antibody comprising the polypeptide of SEQ ID NO:66 and thepolypeptide of SEQ ID NO:82; (m) an antibody comprising the polypeptideof SEQ ID NO:67 and the polypeptide of SEQ ID NO:83; (n) an antibodycomprising the polypeptide of SEQ ID NO:68 and the polypeptide of SEQ IDNO:84; (o) an antibody comprising the polypeptide of SEQ ID NO:69 andthe polypeptide of SEQ ID NO:85; (p) an antibody comprising thepolypeptide of SEQ ID NO:70 and the polypeptide of SEQ ID NO:86; (q) anantibody comprising the polypeptide of SEQ ID NO:71 and the polypeptideof SEQ ID NO:87; and (r) an antibody comprising the polypeptide of SEQID NO:177 and the polypeptide of SEQ ID NO:178.

In certain embodiments, the present disclosure provides a method fortreating a patient having an autoimmune or inflammatory diseasecomprising administering to the patient a therapeutically effectiveamount of an antibody or antigen-binding fragment thereof thatspecifically binds to the same CD37 epitope as an antibody selected fromthe group described above. In some embodiments, the antibody orantigen-binding fragment thereof competitively inhibits an antibodyselected from the group described above.

In certain embodiments, the present disclosure provides a method fordepleting a B-cell comprising contacting a B-cell (e.g., in a populationof cells comprising a non-cancerous B-cell) with an antibody orantigen-binding fragment thereof that specifically binds to CD37 andspecifically binds to the polypeptide of SEQ ID NO: 184. In certainembodiments, the present disclosure provides a method for treating apatient having an autoimmune or inflammatory disease comprisingadministering to the patient a therapeutically effective amount of anantibody or antigen-binding fragment thereof that specifically binds toCD37 and specifically binds to the polypeptide of SEQ ID NO: 184. Insome embodiments, the antibody or antigen-binding fragment thereof doesnot bind to the polypeptide of SEQ ID NO: 185.

In certain embodiments, the present disclosure provides a method fordepleting a B-cell comprising contacting a B-cell (e.g., in a populationof cells comprising a non-cancerous B-cell) with an antibody orantigen-binding fragment thereof that specifically binds to CD37 anddoes not specifically bind to the polypeptide of SEQ ID NO: 185. Incertain embodiments, the present disclosure provides a method fortreating a patient having an autoimmune or inflammatory diseasecomprising administering to the patient a therapeutically effectiveamount of an antibody or antigen-binding fragment thereof thatspecifically binds to CD37 and does not specifically bind to thepolypeptide of SEQ ID NO: 185.

In certain embodiments, the present disclosure provides a method fordepleting a B-cell comprising contacting a B-cell (e.g., in a populationof cells comprising a non-cancerous B-cell) with an antibody orantigen-binding fragment thereof produced by a hybridoma selected fromthe group consisting of ATCC Deposit Designation PTA-10664, depositedwith the ATCC on Feb. 18, 2010, ATCC Deposit Designation PTA-10665,deposited with the ATCC on Feb. 18, 2010, ATCC Deposit DesignationPTA-10666, deposited with the ATCC on Feb. 18, 2010, ATCC DepositDesignation PTA-10667, deposited with the ATCC on Feb. 18, 2010, ATCCDeposit Designation PTA-10668, deposited with the ATCC on Feb. 18, 2010,ATCC Deposit Designation PTA-10669, deposited with the ATCC on Feb. 18,2010, and ATCC Deposit Designation PTA-10670, deposited with the ATCC onFeb. 18, 2010. In certain embodiments, the present disclosure provides amethod for treating a patient having an autoimmune or inflammatorydisease comprising administering to the patient a therapeuticallyeffective amount of an antibody or antigen-binding fragment thereofproduced by a hybridoma described above.

In certain embodiments, the present disclosure provides a method fordepleting a B-cell comprising contacting a B-cell (e.g., in a populationof cells comprising a non-cancerous B-cell) with an antibody orantigen-binding fragment thereof that specifically binds to CD37,wherein the antibody comprises polypeptide sequences selected from thegroup consisting of: (a) SEQ ID NOs: 4, 5, and 6 and SEQ ID NOs: 28, 29,and 30; (b) SEQ ID NOs: 7, 8, and 9 and SEQ ID NOs: 31, 32, and 33; (c)SEQ ID NOs: 10, 11, and 12 and SEQ ID NOs: 34, 35, and 36; (d) SEQ IDNOs: 13, 14, and 15 and SEQ ID NOs: 37, 38, and 39; (e) SEQ ID NOs: 13,14, and 15 and SEQ ID NOs: 37, 40, and 39; (f) SEQ ID NOs: 16, 17, and18 and SEQ ID NOs: 41, 42, and 43; (g) SEQ ID NOs: 19, 20, and 21 andSEQ ID NOs: 44, 45, and 46; (h) SEQ ID NOs: 19, 20, and 21 and SEQ IDNOs: 44, 47, and 46; (i) SEQ ID NOs: 22, 23, and 24 and SEQ ID NOs: 48,49, and 50; (j) SEQ ID NOs: 22, 23, and 24 and SEQ ID NOs: 48, 51, and50; (k) SEQ ID NOs: 25, 26, and 27 and SEQ ID NOs: 52, 53, and 54; (l)SEQ ID NOs: 171, 172 or 181, and 173 and SEQ ID NOs: 174, 175, and 176;(m) variants of (a) to (l) comprising 1, 2, 3, or 4 conservative aminoacid substitutions. In certain embodiments, the present disclosureprovides a method for treating a patient having an autoimmune orinflammatory disease comprising administering to the patient atherapeutically effective amount of an antibody or antigen-bindingfragment thereof with an antibody or antigen-binding fragment thereofthat specifically binds to CD37, wherein the antibody comprisespolypeptide sequences selected from the group described above. In someembodiments, the antibody or antigen-binding fragment thereof comprisespolypeptide sequences that are at least 90% identical to polypeptidesequences described above. In some embodiments, the polypeptidesequences are at least 95% identical to the polypeptide sequences. Insome embodiments, the polypeptide sequences are at least 99% identicalto the polypeptide sequences. In some embodiments, the antibody orantigen-binding fragment thereof comprises polypeptide sequences thatare at least 90% identical, at least 95% identical, at least 99%identical, or identical to the polypeptide sequences of SEQ ID NO: 57and SEQ ID NO:74. In some embodiments, the antibody or antigen-bindingfragment thereof comprises polypeptide sequences that are at least 90%identical, at least 95% identical, at least 99% identical, or identicalto the polypeptide sequences of SEQ ID NO: 58 and SEQ ID NO:74. In someembodiments, the antibody or antigen-binding fragment thereof comprisespolypeptide sequences that are at least 90% identical, at least 95%identical, at least 99% identical, or identical to the polypeptidesequences of SEQ ID NO: 63 and SEQ ID NO:79. In some embodiments, theantibody or antigen-binding fragment thereof comprises polypeptidesequences that are at least 90% identical, at least 95% identical, atleast 99% identical, or identical to the polypeptide sequences of SEQ IDNO: 65 and SEQ ID NO:81.

In some embodiments, the antibody or antigen binding fragment thereof ismurine, non-human, humanized, chimeric, resurfaced, or human.

In some embodiments, the antibody or antibody fragment is capable ofinducing apoptosis of a cell expressing CD37 in vitro in the absence ofcross-linking agents. In some embodiments, the antibody or antigenbinding fragment is capable of inducing complement dependentcytotoxicity (CDC). In some embodiments, the antibody is capable ofinducing antibody dependent cell mediated cytotoxicity (ADCC).

In certain embodiments, the present disclosure provides a method fordepleting a B-cell comprising contacting a B-cell (e.g., in a populationof cells comprising a non-cancerous B-cell) with a human or humanizedantibody or antigen binding fragment thereof that specifically binds toCD37, wherein the antibody or fragment thereof is capable of inducingapoptosis of a cell expressing CD37 in vitro in the absence ofcross-linking agents. In certain embodiments, the present disclosureprovides a method for treating a patient having an autoimmune orinflammatory disease comprising administering to the patient atherapeutically effective amount of a human or humanized antibody orantigen binding fragment thereof that specifically binds to CD37,wherein the antibody or fragment thereof is capable of inducingapoptosis of a cell expressing CD37 in vitro in the absence ofcross-linking agents. In some embodiments, the human or humanizedantibody or antigen binding fragment thereof is also capable of inducingcomplement dependent cytotoxicity (CDC). In some embodiments, the humanor humanized antibody or antigen binding fragment thereof is alsocapable of inducing antibody dependent cell mediated cytotoxicity(ADCC).

In some embodiments, the antibody or antigen-binding fragment binds tohuman CD37 and macaque CD37.

In some embodiments, the antibody is a full length antibody. In someembodiments, an antigen-binding fragment is used. In some embodiments,the antibody or antigen-binding fragment thereof comprises a Fab, Fab′,F(ab′)2, Fd, single chain Fv or scFv, disulfide linked Fv, V-NAR domain,IgNar, intrabody, IgGΔCH2, minibody, F(ab′)3, tetrabody, triabody,diabody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, orscFv-Fc.

In some embodiments, the antibody or antigen-binding fragment thereof islinked via a linker (L) to a cytotoxic agent (C) to form animmunoconjugate.

In certain embodiments, the present disclosure provides a method fordepleting a B-cell comprising contacting a B-cell (e.g., in a populationof cells comprising a non-cancerous B-cell) with a compositioncomprising an immunoconjugate having the formula (A)-(L)-(C), wherein:(A) is an antibody or antigen binding fragment that specifically bindsto CD37; (L) is a non-cleavable linker; and (C) is a cytotoxic agent;and wherein the linker (L) links (A) to (C). In certain embodiments, thepresent disclosure provides a method for treating a patient having anautoimmune or inflammatory disease comprising administering to thepatient a therapeutically effective amount of a composition comprisingan immunoconjugate having the formula (A)-(L)-(C), wherein: (A) is anantibody or antigen binding fragment that specifically binds to CD37;(L) is a non-cleavable linker; and (C) is a cytotoxic agent; and whereinthe linker (L) links (A) to (C). In some embodiments, theimmunoconjugate has a serum half-life that is comparable to that of thenaked antibody.

In certain embodiments, the present disclosure provides a method fordepleting a B-cell comprising contacting a B-cell (e.g., in a populationof cells comprising a non-cancerous B-cell) with a compositioncomprising an immunoconjugate having the formula (A)-(L)-(C), wherein:(A) is an antibody or antigen binding fragment that specifically bindsto CD37; (L) is a linker; and (C) is a maytansinoid; and wherein thelinker (L) links (A) to (C). In certain embodiments, the presentdisclosure provides a method for treating a patient having an autoimmuneor inflammatory disease comprising administering to the patient atherapeutically effective amount of a composition comprising animmunoconjugate having the formula (A)-(L)-(C), wherein: (A) is anantibody or antigen binding fragment that specifically binds to CD37;(L) is a linker; and (C) is a maytansinoid; and wherein the linker (L)links (A) to (C).

In some embodiments, the linker is a non-cleavable linker. In someembodiments, the immunoconjugate further comprises a second (C). In someembodiments, the immunoconjugate further comprises a third (C). In someembodiments, the immunoconjugate further comprises a fourth (C). In someembodiments, the immunoconjugate comprises 2-6 (C). In some embodiments,the immunoconjugate comprises 3-4 (C).

In some embodiments, the linker is selected from the group consisting ofa cleavable linker, a non-cleavable linker, a hydrophilic linker, and adicarboxylic acid based linker. In some embodiments, the linker isselected from the group consisting of: N-succinimidyl4-(2-pyridyldithio)pentanoate (SPP); N-succinimidyl4-(2-pyridyldithio)butanoate (SPDB) or N-succinimidyl4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB); N-succinimidyl4-(maleimidomethyl) cyclohexanecarboxylate (SMCC); N-sulfosuccinimidyl4-(maleimidomethyl) cyclohexanecarboxylate (sulfoSMCC);N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB); andN-succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol]ester(NHS-PEG4-maleimide). In some embodiments, the linker isN-succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol] ester(NHS-PEG4-maleimide).

In some embodiments, the cytotoxic agent is selected from the groupconsisting of a maytansinoid, maytansinoid analog, doxorubicin, amodified doxorubicin, benzodiazepine, taxoid, CC-1065, CC-1065 analog,duocarmycin, duocarmycin analog, calicheamicin, dolastatin, dolastatinanalog, auristatin, tomaymycin derivative, and leptomycin derivative ora prodrug of the agent. In some embodiments, the cytotoxic agent is amaytansinoid. In some embodiments, the cytotoxic agent isN(2′)-deacetyl-N(2′)-(3-mercapto-1-oxopropyl)-maytansine (DM1) orN(2)-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4).

In some embodiments, the composition comprising an immunoconjugatecomprises multiple cytotoxic agents (C) with an average of about 3 toabout 4 (C) per (A). In some embodiments, the immunoconjugates have anaverage of about 3.5 (C) per (A). In some embodiments, theimmunoconjugates have an average of about 3.5±0.5 (C) per (A).

In some embodiments, the composition comprising an immunoconjugatecomprises an antibody comprising SEQ ID NO:57 and SEQ ID NO:74 or SEQ IDNO:58 and SEQ ID NO:74, an SMCC linker, and DM1. In some embodiments,the composition comprising an immunoconjugate comprises an antibodycomprising SEQ ID NO:63 and SEQ ID NO:79, an SMCC linker, and DM1. Insome embodiments, the composition comprising an immunoconjugatecomprises an antibody comprising SEQ ID NO:65 and SEQ ID NO:81, an SMCClinker, and DM1.

In some embodiments, the antibody or antigen-binding fragment is capableof depleting B-cells. In some embodiments, the antibody orantigen-binding fragment is capable of inhibiting T-cell responses.

In some embodiments, the B-cell is in a composition further comprising aT-cell. In some embodiments, the B-cell is in a composition comprisingperipheral blood mononuclear cells. In some embodiments, the peripheralblood mononuclear cells were obtained from a human. In some embodiments,the B-cell is in whole blood. In some embodiments, the whole blood wasobtained from a human. In some embodiments, the B-cell is in anorganism. In some embodiments, the B-cell is in a patient having anautoimmune or inflammatory disease.

In some embodiments, the B-cell is an autoreactive B-cell.

In some embodiments, at least about 30% of B-cells are depleted. In someembodiments, less than about 5% of T-cells are depleted.

In some embodiments, a second therapeutic agent is administered. In someembodiments, the second therapeutic is selected from the groupconsisting of methotrexate, an anti-CD20 therapeutic, an anti-IL-6receptor therapeutic, an anti-IL-12/23p40 therapeutic, achemotherapeutic, an immunosuppressant, an anti-Interferon beta-1atherapeutic, glatiramer acetate, an anti-α4-integrin therapeutic,fingolimod, an anti-BLys therapeutic, CTLA-Fc, or an anti-TNFtherapeutic. In some embodiments, the second therapeutic is an antibodydirected against an antigen selected from a group consisting of CD3,CD14, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD36, CD38, CD40, CD44,CD52, CD55, CD59, CD56, CD70, CD79, CD80, CD103, CD134, CD137, CD138,and CD152. In some embodiments, the second therapeutic is an antibodydirected against an antigen selected from the group consisting of IL-2,IL-6, IL-12, IL-23, IL-12/23 p40, IL-17, IFNγ, TNFα, IFNα, IL-15, IL-21,IL-1a, IL-1b, IL-18, IL-8, IL-4, GM-CSF, IL-3, and IL-5.

In some embodiments, the autoimmune or inflammatory disease is selectedfrom the group consisting of rheumatoid arthritis, multiple sclerosis,type I diabetes mellitus, idiopathic inflammatory myopathy, systemiclupus erythematosus (SLE), myasthenia gravis, Grave's disease,dermatomyositis, polymyositis, Crohn's disease, ulcerative colitis,gastritis, Hashimoto's thyroiditis, asthma, psoriasis, psoriaticarthritis, dermatitis, systemic scleroderma and sclerosis, inflammatorybowel disease (IBD), respiratory distress syndrome, meningitis,encephalitis, uveitis, glomerulonephritis, eczema, atherosclerosis,leukocyte adhesion deficiency, Raynaud's syndrome, Sjögen's syndrome,Reiter's disease, Beheet's disease, immune complex nephritis, IgAnephropathy, IgM polyneuropathies, immune-mediated thrombocytopenias,acute idiopathic thrombocytopenic purpura, chronic idiopathicthembocytopenic purpura, hemolytic anemia, myasthenia gravis, lupusnephritis, atopic dermatitis, pemphigus vulgaris, opsoclonus-myoclonussyndrome, pure red cell aplasia, mixed cryoglobulinermia, ankylosingspondylitis, hepatitis C-associated cryoglobulinemic vasculitis, chronicfocal encephalitis, bullous pemphigoid, hemophilia A,membranoproliferative glomerulonephritis, adult and juveniledermatomyositis, adult polymyositis, chronic urticaria, primary biliarycirrhosis, neuromyelitis optica, Graves' dysthyroid disease, bullouspemphigoid, membranoproliferative glomerulonephritis, Churg-Strausssyndrome, juvenile onset diabetes, hemolytic anemia, atopic dermatitis,systemic sclerosis, Sjögen's syndrome and glomerulonephritis,dermatomyositis, ANCA, aplastic anemia, autoimmune hemolytic anemia(AIHA), factor VIII deficiency, hemophilia A, autoimmune neutropenia,Castleman's syndrome, Goodpasture's syndrome, solid organ transplantrejection, graft versus host disease (GVHD), autoimmune hepatitis,lymphoid interstitial pneumonitis, HIV, bronchiolitis obliterans(non-transplant), Guillain-Barre Syndrome, large vessel vasculitis,giant cell (Takayasu's) arteritis, medium vessel vasculitis, Kawasaki'sDisease, polyarteritis nodosa. Wegener's granulomatosis, microscopicpolyangiitis (MPA), Omenn's syndrome, chronic renal failure, acuteinfectious mononucleosis, HIV and herpes virus associated diseases.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 depicts an FL2-H (PE) histogram overlay for a flow cytometryexperiment with human B-cells. The following conditions are shown:antibody control (dark filled), isotype control stain (light filled),anti-CD37 stain (thick black line), and anti-CD20 stain (dashed line)for CD19+ B-cells.

FIG. 2 depicts the results of in vitro depletion experiments usingpurified human PBMC samples treated with 10 μg/mL of huCD37-3,huCD37-3-SMCC-DM1, huCD37-50, huCD37-50-SMCC-DM1, rituximab, TRU-016, oralemtuzumab. Results from two different donors are shown in panel A andB.

FIG. 3 depicts the results of in vitro depletion experiments usingpurified human PBMC samples treated with varying concentrations ofhuCD37-3-SMCC-DM1. Results from two different donors are shown in panelsA and B. FIG. 3 (C) shows the results using huCD37-3, huCD37-38,huCD37-50 and huCD37-56.

FIG. 4 depicts the results of in vitro depletion experiments usingunpurified whole human blood samples treated with 10 μg/mL of huCD37-3,huCD37-3-SMCC-DM1, huCD37-50, huCD37-50-SMCC-DM1, rituximab, TRU-016, oralemtuzumab.

FIG. 5 depicts the results of in vitro depletion experiments usingunpurified whole human blood samples treated with varying concentrationsof (A) huCD37-3, huCD37-3-SMCC-DM1, and rituximab and (B) huCD37-3,huCD37-3-SMCC-DM1, huCD37-50, and rituximab.

FIG. 6 depicts release of IFN-γ (Interferon), TNF-α (Tumor NecrosisFactor) and IL-6 (Interleukin-6) measured by ELISpot as number of spotsper 5×10E5 peripheral blood mononuclear cells (PBMCs) from one healthyhuman donor incubated for 18-20 hours with compounds at a concentrationof 2.5 ng/mL to 250 μg/mL.

FIG. 7 depicts release of IFN-γ (Interferon), TNF-α (Tumor NecrosisFactor) and IL-6 (Interleukin-6) measured by ELISpot as number of spotsper 5×10⁵ peripheral blood mononuclear cells (PBMCs) from a secondhealthy human donor incubated for 18-20 hours with compounds at aconcentration of 2.5 ng/mL to 250 μg/mL.

FIG. 8 depicts the binding curve of anti-muCD37 monoclonal antibodyclone 252-3.

FIG. 9 shows the activity of the 252-3 antibody in depleting peripheralblood B cells (A) and in inhibiting EAE (B) in C57Bl/6 mice. In (A),each symbol represent one mouse; to compare the B cell level in controlvs. experimental mice, B cell level was normalized with T cell level andratio of B/T cell in control mice was considered 100%. In (B), open andclosed symbols represent mean of EAE score in control group (n=10) and252-3 antibody treated group (n=10), respectively; arrow indicates dayof antibody injection.

FIG. 10 shows the activity of the 252-3 antibody in depleting peripheralblood B cells (A) and in inhibiting T1D (B) in NOD mice. In (A), eachsymbol represent one mouse; to compare the B cell level in control vs.experimental mice, B cell level was normalized with T cell level andratio of B/T cell in control mice was considered 100%. In (B), open andclosed symbols represent the diabetes incidence in control group (n=6)and 252-3 antibody treated group (n=6), respectively.

FIG. 11 shows the activity of the 252-3 antibody in depleting peripheralblood B cells (A) and in inhibiting CIA (B) in DBA/1 mice. In (A), eachsymbol represent one mouse; to compare the B cell level in control vs.experimental mice, B cell level was normalized with T cell level andratio of B/T cell in control mice was considered 100%. In (B), open andclosed symbols represents mean of CIA score in control group (n=12) and252-3 antibody treated group (n=12), respectively; arrow indicates dayof antibody injection.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of depleting B-cells and oftreating diseases associated with aberrant B-cell activity and/oraberrant T-cell stimulation in connection with a B-cell pathway usingCD37 binding molecules.

I. Definitions

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below.

The term CD37 as used herein, refers to any native CD37, unlessotherwise indicated. CD37 is also referred to as GP52-40, leukocyteantigen CD37, and Tetraspanin-26. The term “CD37” encompasses“full-length,” unprocessed CD37 as well as any form of CD37 that resultsfrom processing in the cell. The term also encompasses naturallyoccurring variants of CD37, e.g., splice variants, allelic variants, andisoforms. The CD37 polypeptides described herein can be isolated from avariety of sources, such as from human tissue types or from anothersource, or prepared by recombinant or synthetic methods.

The term “antibody” means an immunoglobulin molecule that recognizes andspecifically binds to a target, such as a protein, polypeptide, peptide,carbohydrate, polynucleotide, lipid, or combinations of the foregoingthrough at least one antigen recognition site within the variable regionof the immunoglobulin molecule. As used herein, the term “antibody”encompasses intact polyclonal antibodies, intact monoclonal antibodies,antibody fragments (such as Fab, Fab′, F(ab′)2, and Fv fragments),single chain Fv (scFv) mutants, multispecific antibodies such asbispecific antibodies generated from at least two intact antibodies,chimeric antibodies, humanized antibodies, human antibodies, fusionproteins comprising an antigen determination portion of an antibody, andany other modified immunoglobulin molecule comprising an antigenrecognition site so long as the antibodies exhibit the desiredbiological activity. An antibody can be of any the five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes)thereof (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on theidentity of their heavy-chain constant domains referred to as alpha,delta, epsilon, gamma, and mu, respectively. The different classes ofimmunoglobulins have different and well known subunit structures andthree-dimensional configurations. Antibodies can be naked or conjugatedto other molecules such as toxins, radioisotopes, etc.

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds, such as CD37. Insome embodiments, blocking antibodies or antagonist antibodiessubstantially or completely inhibit the biological activity of theantigen. The biological activity can be reduced by 10%, 20%, 30%, 50%,70%, 80%, 90%, 95%, or even 100%.

The term “anti-CD37 antibody” or “an antibody that binds to CD37” refersto an antibody that is capable of binding CD37 with sufficient affinitysuch that the antibody is useful as a diagnostic and/or therapeuticagent in targeting CD37. The extent of binding of an anti-CD37 antibodyto an unrelated, non-CD37 protein can be less than about 10% of thebinding of the antibody to CD37 as measured, e.g., by a radioimmunoassay(RIA). In certain embodiments, an antibody that binds to CD37 has adissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM.

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limited toFab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, single chainantibodies, and multispecific antibodies formed from antibody fragments.

A “monoclonal antibody” refers to a homogeneous antibody populationinvolved in the highly specific recognition and binding of a singleantigenic determinant, or epitope. This is in contrast to polyclonalantibodies that typically include different antibodies directed againstdifferent antigenic determinants. The term “monoclonal antibody”encompasses both intact and full-length monoclonal antibodies as well asantibody fragments (such as Fab, Fab′, F(ab′)2, Fv), single chain (scFv)mutants, fusion proteins comprising an antibody portion, and any othermodified immunoglobulin molecule comprising an antigen recognition site.Furthermore, “monoclonal antibody” refers to such antibodies made in anynumber of manners including but not limited to by hybridoma, phageselection, recombinant expression, and transgenic animals.

The term “humanized antibody” refers to forms of non-human (e.g. murine)antibodies that are specific immunoglobulin chains, chimericimmunoglobulins, or fragments thereof that contain minimal non-human(e.g., murine) sequences. Typically, humanized antibodies are humanimmunoglobulins in which residues from the complementary determiningregion (CDR) are replaced by residues from the CDR of a non-humanspecies (e.g. mouse, rat, rabbit, hamster) that have the desiredspecificity, affinity, and capability (Jones et al., 1986, Nature,321:522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen etal., 1988, Science, 239:1534-1536). In some instances, the Fv frameworkregion (FR) residues of a human immunoglobulin are replaced with thecorresponding residues in an antibody from a non-human species that hasthe desired specificity, affinity, and capability. The humanizedantibody can be further modified by the substitution of additionalresidues either in the Fv framework region and/or within the replacednon-human residues to refine and optimize antibody specificity,affinity, and/or capability. In general, the humanized antibody willcomprise substantially all of at least one, and typically two or three,variable domains containing all or substantially all of the CDR regionsthat correspond to the non-human immunoglobulin whereas all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody can also comprise at least aportion of an immunoglobulin constant region or domain (Fc), typicallythat of a human immunoglobulin. Examples of methods used to generatehumanized antibodies are described in U.S. Pat. No. 5,225,539.

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination. The variable regions of the heavy andlight chain each consist of four framework regions (FR) connected bythree complementarity determining regions (CDRs) also known ashypervariable regions. The CDRs in each chain are held together in closeproximity by the FRs and, with the CDRs from the other chain, contributeto the formation of the antigen-binding site of antibodies. There are atleast two techniques for determining CDRs: (1) an approach based oncross-species sequence variability (i.e., Kabat et al. Sequences ofProteins of Immunological Interest, (5th ed., 1991, National Institutesof Health, Bethesda Md.)); and (2) an approach based on crystallographicstudies of antigen-antibody complexes (Al-lazikani et al (1997) J.Molec. Biol. 273:927-948)). In addition, combinations of these twoapproaches are sometimes used in the art to determine CDRs.

The Kabat numbering system is generally used when referring to a residuein the variable domain (approximately residues 1-107 of the light chainand residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences ofImmunological Interest. 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)).

The amino acid position numbering as in Kabat, refers to the numberingsystem used for heavy chain variable domains or light chain variabledomains of the compilation of antibodies in Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991). Using thisnumbering system, the actual linear amino acid sequence can containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FR or CDR of the variable domain. For example, a heavychain variable domain can include a single amino acid insert (residue52a according to Kabat) after residue 52 of H2 and inserted residues(e.g. residues 82a, 82b, and 82c, etc., according to Kabat) after heavychain FR residue 82. The Kabat numbering of residues can be determinedfor a given antibody by alignment at regions of homology of the sequenceof the antibody with a “standard” Kabat numbered sequence. Chothiarefers instead to the location of the structural loops (Chothia and LeskJ. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loopwhen numbered using the Kabat numbering convention varies between H32and H34 depending on the length of the loop (this is because the Kabatnumbering scheme places the insertions at H35A and H35B; if neither 35Anor 35B is present, the loop ends at 32; if only 35A is present, theloop ends at 33; if both 35A and 35B are present, the loop ends at 34).The AbM hypervariable regions represent a compromise between the KabatCDRs and Chothia structural loops, and are used by Oxford Molecular'sAbM antibody modeling software.

Loop Kabat AbM Chothia L1 L24-L34 L24-L34 L24-L34 L2 L50-L56 L50-L56L50-L56 L3 L89-L97 L89-L97 L89-L97 H1 H31-H35B H26-H35B H26-H32 . . . 34(Kabat Numbering) H1 H31-H35 H26-H35 H26-H32 (Chothia Numbering) H2H50-H65 H50-E58 H52-H56 H3 H95-H102 H95-H102 H95-H102

The term “human antibody” means an antibody produced by a human or anantibody having an amino acid sequence corresponding to an antibodyproduced by a human made using any technique known in the art. Thisdefinition of a human antibody includes intact or full-lengthantibodies, fragments thereof, and/or antibodies comprising at least onehuman heavy and/or light chain polypeptide such as, for example, anantibody comprising murine light chain and human heavy chainpolypeptides.

The term “chimeric antibodies” refers to antibodies wherein the aminoacid sequence of the immunoglobulin molecule is derived from two or morespecies. Typically, the variable region of both light and heavy chainscorresponds to the variable region of antibodies derived from onespecies of mammals (e.g. mouse, rat, rabbit, etc.) with the desiredspecificity, affinity, and capability while the constant regions arehomologous to the sequences in antibodies derived from another (usuallyhuman) to avoid eliciting an immune response in that species.

The term “epitope” or “antigenic determinant” are used interchangeablyherein and refer to that portion of an antigen capable of beingrecognized and specifically bound by a particular antibody. When theantigen is a polypeptide, epitopes can be formed both from contiguousamino acids and noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids are typicallyretained upon protein denaturing, whereas epitopes formed by tertiaryfolding are typically lost upon protein denaturing. An epitope typicallyincludes at least 3, and more usually, at least 5 or 8-10 amino acids ina unique spatial conformation.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthe present invention. Specific illustrative embodiments are describedin the following.

“Or better” when used herein to refer to binding affinity refers to astronger binding between a molecule and its binding partner. “Or better”when used herein refers to a stronger binding, represented by a smallernumerical Kd value. For example, an antibody which has an affinity foran antigen of “0.6 nM or better”, the antibody's affinity for theantigen is <0.6 nM, i.e. 0.59 nM, 0.58 nM, 0.57 nM etc. or any valueless than 0.6 nM.

By “specifically binds,” it is generally meant that an antibody binds toan epitope via its antigen binding domain, and that the binding entailssome complementarity between the antigen binding domain and the epitope.According to this definition, an antibody is said to “specifically bind”to an epitope when it binds to that epitope, via its antigen bindingdomain more readily than it would bind to a random, unrelated epitope.The term “specificity” is used herein to qualify the relative affinityby which a certain antibody binds to a certain epitope. For example,antibody “A” may be deemed to have a higher specificity for a givenepitope than antibody “B,” or antibody “A” may be said to bind toepitope “C” with a higher specificity than it has for related epitope“D.”

By “preferentially binds,” it is meant that the antibody specificallybinds to an epitope more readily than it would bind to a related,similar, homologous, or analogous epitope. Thus, an antibody which“preferentially binds” to a given epitope would more likely bind to thatepitope than to a related epitope, even though such an antibody maycross-react with the related epitope.

An antibody is said to “competitively inhibit” binding of a referenceantibody to a given epitope if it preferentially binds to that epitopeto the extent that it blocks, to some degree, binding of the referenceantibody to the epitope. Competitive inhibition may be determined by anymethod known in the art, for example, competition ELISA assays. Anantibody may be said to competitively inhibit binding of the referenceantibody to a given epitope by at least 90%, at least 80%, at least 70%,at least 60%, or at least 50%.

The phrase “substantially similar,” or “substantially the same”, as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (generally one associated with an antibody of theinvention and the other associated with a reference/comparator antibody)such that one of skill in the art would consider the difference betweenthe two values to be of little or no biological and/or statisticalsignificance within the context of the biological characteristicmeasured by said values (e.g., Kd values). The difference between saidtwo values can be less than about 50%, less than about 40%, less thanabout 30%, less than about 20%, or less than about 10% as a function ofthe value for the reference/comparator antibody.

A polypeptide, antibody, polynucleotide, vector, cell, or compositionwhich is “isolated” is a polypeptide, antibody, polynucleotide, vector,cell, or composition which is in a form not found in nature. Isolatedpolypeptides, antibodies, polynucleotides, vectors, cell or compositionsinclude those which have been purified to a degree that they are nolonger in a form in which they are found in nature. In some embodiments,an antibody, polynucleotide, vector, cell, or composition which isisolated is substantially pure.

As used herein, “substantially pure” refers to material which is atleast 50% pure (i.e., free from contaminants), at least 90% pure, atleast 95% pure, at least 98% pure, or at least 99% pure.

The term “immunoconjugate” or “conjugate” as used herein refers to acompound or a derivative thereof that is linked to a cell binding agent(i.e., an anti-CD37 antibody or fragment thereof) and is defined by ageneric formula: C-L-A, wherein C=cytotoxin, L=linker, and A=cellbinding agent or anti-CD37 antibody or antibody fragment.Immunoconjugates can also be defined by the generic formula in reverseorder: A-L-C.

A “linker” is any chemical moiety that is capable of linking a compound,usually a drug, such as a maytansinoid, to a cell-binding agent such asan anti CD37 antibody or a fragment thereof in a stable, covalentmanner. Linkers can be susceptible to or be substantially resistant toacid-induced cleavage, light-induced cleavage, peptidase-inducedcleavage, esterase-induced cleavage, and disulfide bond cleavage, atconditions under which the compound or the antibody remains active.Suitable linkers are well known in the art and include, for example,disulfide groups, thioether groups, acid labile groups, photolabilegroups, peptidase labile groups and esterase labile groups. Linkers alsoinclude charged linkers, and hydrophilic forms thereof as describedherein and know in the art.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals in which a population of cells arecharacterized by unregulated cell growth. Examples of cancer include,but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia. “Tumor” and “neoplasm” refer to one or more cells that resultfrom excessive cell growth or proliferation, either benign(noncancerous) or malignant (cancerous) including pre-cancerous lesions.Examples of “cancer” or “tumorigenic” diseases which can be treatedand/or prevented include B-cell lymphomas including NHL, precursorB-cell lymphoblastic leukemia/lymphoma and mature B-cell neoplasms, suchas B-cell chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma(SLL), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma,mantle cell lymphoma (MCL), follicular lymphoma (FL), includinglow-grade, intermediate-grade and high-grade FL, cutaneous folliclecenter lymphoma, marginal zone B-cell lymphoma (MALT type, nodal andsplenic type), hairy cell leukemia, diffuse large B-cell lymphoma,Burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplantlymphoproliferative disorder, and anaplastic large-cell lymphoma (ALCL).Non-cancerous cells are cells that do not result in the formation oftumors or neoplasms or the development of cancer. However, non-cancerouscells can contribute to disease, e.g., autoimmune diseases, and include,for example auto-reactive B-cells.

The terms “cancer cell,” “tumor cell,” and grammatical equivalents referto the total population of cells derived from a tumor or a pre-cancerouslesion, including both non-tumorigenic cells, which comprise the bulk ofthe tumor cell population, and tumorigenic stem cells (cancer stemcells). As used herein, the term “tumor cell” will be modified by theterm “non-tumorigenic” when referring solely to those tumor cellslacking the capacity to renew and differentiate to distinguish thosetumor cells from cancer stem cells.

The term “autoreactive” refers to a cell, tissue, protein, antibody orother substance that produces an immune response directed against anorganism's own cells, tissues, proteins, antibodies, or othersubstances.

The term “subject” refers to any animal (e.g., a mammal), including, butnot limited to humans, non-human primates, rodents, and the like, whichis to be the recipient of a particular treatment. Typically, the terms“subject” and “patient” are used interchangeably herein in reference toa human subject.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of the activeingredient to be effective, and which contains no additional componentswhich are unacceptably toxic to a subject to which the formulation wouldbe administered. The formulation can be sterile.

An “effective amount” of an antibody as disclosed herein is an amountsufficient to carry out a specifically stated purpose. An “effectiveamount” can be determined empirically and in a routine manner, inrelation to the stated purpose.

The term “therapeutically effective amount” refers to an amount of anantibody or other drug effective to “treat” a disease or disorder in asubject or mammal. In some embodiments, the therapeutically effectiveamount of the drug can reduce the number of B-cells; reduce the numberof autoreactive B-cells; decrease the symptoms of disease; or slow theprogression of disease. See the definition herein of “treating”. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically but not necessarily, since a prophylacticdose is used in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibodyso as to generate a “labeled” antibody. The label can be detectable byitself (e.g., radioisotope labels or fluorescent labels) or, in the caseof an enzymatic label, can catalyze chemical alteration of a substratecompound or composition which is detectable.

Terms such as “treating” or “treatment” or “to treat” or “alleviating”or “to alleviate” refer to therapeutic measures that cure, slow down,lessen symptoms of, and/or halt progression of a diagnosed pathologiccondition or disorder. Thus, those in need of treatment include thosealready diagnosed with or suspected of having the disorder. Prophylacticor preventative measures refer to therapeutic measures that preventand/or slow the development of a targeted pathologic condition ordisorder. Thus, those in need of prophylactic or preventative measuresinclude those prone to have the disorder and those in whom the disorderis to be prevented. In certain embodiments, a subject is successfully“treated” if the patient shows one or more of the following: decreasedB-cells; decreased autoreactive B-cells; decreased B-cell activity;decreased aberrant B-cell activity; decreased non-malignant B-cells,decreased non-cancerous B-cells, reduced immunoglobulin level; reducedmorbidity and mortality; improvement in quality of life; or somecombination of effects.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase. A polynucleotidecan comprise modified nucleotides, such as methylated nucleotides andtheir analogs. If present, modification to the nucleotide structure canbe imparted before or after assembly of the polymer. The sequence ofnucleotides can be interrupted by non-nucleotide components. Apolynucleotide can be further modified after polymerization, such as byconjugation with a labeling component. Other types of modificationsinclude, for example, “caps”, substitution of one or more of thenaturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotide(s). Further, any ofthe hydroxyl groups ordinarily present in the sugars can be replaced,for example, by phosphonate groups, phosphate groups, protected bystandard protecting groups, or activated to prepare additional linkagesto additional nucleotides, or can be conjugated to solid supports. The5′ and 3′ terminal OH can be phosphorylated or substituted with aminesor organic capping group moieties of from 1 to 20 carbon atoms. Otherhydroxyls can also be derivatized to standard protecting groups.Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, forexample, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose,carbocyclic sugar analogs, .alpha.-anomeric sugars, epimeric sugars suchas arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs and abasic nucleoside analogs such asmethyl riboside. One or more phosphodiester linkages can be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S (“thioate”), P(S)S (“dithioate”), “(O)NR₂ (“amidate”), P(O)R,P(O)OR′, CO or CH₂ (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl,cycloalkenyl or araldyl. Not all linkages in a polynucleotide need beidentical. The preceding description applies to all polynucleotidesreferred to herein, including RNA and DNA.

The term “vector” means a construct, which is capable of delivering, andoptionally expressing, one or more gene(s) or sequence(s) of interest ina host cell. Examples of vectors include, but are not limited to, viralvectors, naked DNA or RNA expression vectors, plasmid, cosmid or phagevectors, DNA or RNA expression vectors associated with cationiccondensing agents, DNA or RNA expression vectors encapsulated inliposomes, and certain eukaryotic cells, such as producer cells.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer can be linear or branched, it can comprise modifiedamino acids, and it can be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art. Itis understood that, because the polypeptides of this invention are basedupon antibodies, in certain embodiments, the polypeptides can occur assingle chains or associated chains.

The terms “identical” or percent “identity” in the context of two ormore nucleic acids or polypeptides, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides or amino acid residues that are the same, when compared andaligned (introducing gaps, if necessary) for maximum correspondence, notconsidering any conservative amino acid substitutions as part of thesequence identity. The percent identity can be measured using sequencecomparison software or algorithms or by visual inspection. Variousalgorithms and software are known in the art that can be used to obtainalignments of amino acid or nucleotide sequences. One such non-limitingexample of a sequence alignment algorithm is the algorithm described inKarlin et al, 1990, Proc. Natl. Acad. Sci., 87:2264-2268, as modified inKarlin et al., 1993, Proc. Natl. Acad. Sci., 90:5873-5877, andincorporated into the NBLAST and XBLAST programs (Altschul et al., 1991,Nucleic Acids Res., 25:3389-3402). In certain embodiments, Gapped BLASTcan be used as described in Altschul et al., 1997, Nucleic Acids Res.25:3389-3402. BLAST-2, WU-BLAST-2 (Altschul et al., 1996, Methods inEnzymology, 266:460-480), ALIGN, ALIGN-2 (Genentech, South SanFrancisco, Calif.) or Megalign (DNASTAR) are additional publiclyavailable software programs that can be used to align sequences. Incertain embodiments, the percent identity between two nucleotidesequences is determined using the GAP program in GCG software (e.g.,using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90and a length weight of 1, 2, 3, 4, 5, or 6). In certain alternativeembodiments, the GAP program in the GCG software package, whichincorporates the algorithm of Needleman and Wunsch (J. Mol. Biol.(48):444-453 (1970)) can be used to determine the percent identitybetween two amino acid sequences (e.g., using either a Blossum 62 matrixor a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and alength weight of 1, 2, 3, 4, 5). Alternatively, in certain embodiments,the percent identity between nucleotide or amino acid sequences isdetermined using the algorithm of Myers and Miller (CABIOS, 4:11-17(1989)). For example, the percent identity can be determined using theALIGN program (version 2.0) and using a PAM120 with residue table, a gaplength penalty of 12 and a gap penalty of 4. Appropriate parameters formaximal alignment by particular alignment software can be determined byone skilled in the art. In certain embodiments, the default parametersof the alignment software are used. In certain embodiments, thepercentage identity “X” of a first amino acid sequence to a secondsequence amino acid is calculated as 100×(Y/Z), where Y is the number ofamino acid residues scored as identical matches in the alignment of thefirst and second sequences (as aligned by visual inspection or aparticular sequence alignment program) and Z is the total number ofresidues in the second sequence. If the length of a first sequence islonger than the second sequence, the percent identity of the firstsequence to the second sequence will be longer than the percent identityof the second sequence to the first sequence.

As a non-limiting example, whether any particular polynucleotide has acertain percentage sequence identity (e.g., is at least 80% identical,at least 85% identical, at least 90% identical, and in some embodiments,at least 95%, 96%, 97%, 98%, or 99% identical) to a reference sequencecan, in certain embodiments, be determined using the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). Bestfit uses the local homology algorithm of Smith andWaterman, Advances in Applied Mathematics 2: 482 489 (1981), to find thebest segment of homology between two sequences. When using Bestfit orany other sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set such that thepercentage of identity is calculated over the full length of thereference nucleotide sequence and that gaps in homology of up to 5% ofthe total number of nucleotides in the reference sequence are allowed.

In some embodiments, two nucleic acids or polypeptides of the inventionare substantially identical, meaning they have at least 70%, at least75%, at least 80%, at least 85%, at least 90%, and in some embodimentsat least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residueidentity, when compared and aligned for maximum correspondence, asmeasured using a sequence comparison algorithm or by visual inspection.Identity can exist over a region of the sequences that is at least about10, about 20, about 40-60 residues in length or any integral valuetherebetween, and can be over a longer region than 60-80 residues, forexample, at least about 90-100 residues, and in some embodiments, thesequences are substantially identical over the full length of thesequences being compared, such as the coding region of a nucleotidesequence for example.

A “conservative amino acid substitution” is one in which one amino acidresidue is replaced with another amino acid residue having a similarside chain Families of amino acid residues having similar side chainshave been defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). For example, substitution of aphenylalanine for a tyrosine is a conservative substitution. In someembodiments, conservative substitutions in the sequences of thepolypeptides and antibodies of the invention do not abrogate the bindingof the polypeptide or antibody containing the amino acid sequence, tothe antigen(s), i.e., the CD37 to which the polypeptide or antibodybinds. Methods of identifying nucleotide and amino acid conservativesubstitutions which do not eliminate antigen binding are well-known inthe art (see, e.g., Brummell et al., Biochem. 32: 1180-1 187 (1993);Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al.Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

As used in the present disclosure and claims, the singular forms “a,”“an,” and “the” include plural forms unless the context clearly dictatesotherwise.

It is understood that wherever embodiments are described herein with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

The term “and/or” as used in a phrase such as “A and/or B” herein isintended to include both “A and B,” “A or B,” “A,” and “B.” Likewise,the term “and/or” as used in a phrase such as “A, B, and/or C” isintended to encompass each of the following embodiments: A, B, and C; A,B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B(alone); and C (alone).

II. CD37 Binding Agents

The present invention provides agents that specifically bind CD37. Theseagents are referred to herein as “CD37 binding agents.” ExemplaryCD37-binding agents have been described in U.S. Published ApplicationNo. 2011/0256153, which is herein incorporated by reference in itsentirety.

The full-length amino acid sequences for human, macaca, and murine CD37are known in the art and also provided herein as represented by SEQ IDNOs:1-3, respectively.

Human CD37: (SEQ ID NO: 1)MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKVLAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRAQLERSLRDVVEKTIQKYGTNPEETAAEESWDYVQFQLRCCGWHYPQDWFQVLILRGNGSEAHRVPCSCYNLSATNDSTILDKVILPQLSRLGHLARSRHSADICAVPAESHIYREGCAQGLQKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLAYR Macaca mulatta CD37: (SEQ ID NO: 2)MSAQESCLSLIKYFLFVFNLFFFVILGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKVLAISGVFTMGLALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRAQLERSLQDIVEKTIQRYHTNPEETAAEESWDYVQFQLRCCGWHSPQDWFQVLTLRGNGSEAHRVPCSCYNLSATNDSTILDKVILPQLSRLGQLARSRHSTDICAVPANSHIYREGCARSLQKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLRYR Murine CD37 (NP_031671): (SEQ ID NO: 3)MSAQESCLSLIKYFLFVFNLFFFVLGGLIFCFGTWILIDKTSFVSFVGLSFVPLQTWSKVLAVSGVLTMALALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRVRLERRVQELVLRTIQSYRTNPDETAAEESWDYAQFQLRCCGWQSPRDWNKAQMLKANESEEPFVPCSCYNSTATNDSTVFDKLFFSQLSRLGPRAKLRQTADICALPAKAHIYREGCAQSLQKWLHNNIISIVGICLGVGLLELGFMTLSIFLCRNLDHVYDRLARYR

In certain embodiments, the CD37 binding agents are antibodies,immunoconjugates or polypeptides. In some embodiments, the CD37 bindingagents are humanized antibodies.

In certain embodiments, the CD37-binding agents are capable of inducingcomplement dependent cytotoxicity. Examples of CD37-binding agents thatare capable of inducing complement dependent cytotoxicity are disclosed,for example, in U.S. Published Application No. 2011/0256153, which isherein incorporated by reference in its entirety. For example, treatmentof cells with the CD37-binding agents can result in CDC activity thatreduces cell viability to less than about 80%, less than about 70%, lessthan about 60%, less than about 50%, less than about 40% or less thanabout 35% of the cell viability of untreated cells. Treatment of cellswith the CD37-binding agents can also result in CDC activity thatreduces cell viability to about 70-80%, about 60-70%, about 50-60%,about 40-50%, or about 30-40% of the cell viability of untreated cells.In some particular embodiments, the CD37-binding agents are capable ofinducing complement dependent cytotoxicity in Ramos cells.

In certain embodiments, the CD37-binding agents are capable of inducingantibody dependent cell mediated cytotoxicity (ADCC). Examples of CD-37binding agents that are capable of inducing antibody dependent cellmediated cytotoxicity (ADCC) are disclosed, for example, in U.S.Published Application No. 2011/0256153, which is herein incorporated byreference in its entirety. For example, treatment of cells with theCD37-binding agents can result in ADCC activity that produces at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, or at least about 60% cell lysis. Treatment of cells with theCD37-binding agents can result in ADCC activity that produces about10-20%, about 20-30%, about 30-40%, or about 40-50% cell lysis.Treatment of cells with the CD37-binding agents can also result in ADCCactivity that produces about 10-50%, about 20-50%, about 30-50%, orabout 40-50% cell lysis. In some particular embodiments, theCD37-binding agents are capable of inducing ADCC in Daudi, Ramos, and/orGranata-519 cells.

In some embodiments, the CD37-binding agents are capable of inducingapoptosis. In some embodiment, the CD37-binding agents are capable ofinducing apoptosis in the absence of cross-linking agents. Examples ofCD37-binding agents that are capable of inducing apoptosis in vitro inthe absence of a cross-linking agent are disclosed, for example, in U.S.Published Application No. 2011/0256153, which is herein incorporated byreference in its entirety. For example, treatment of cells with theCD37-binding agents can induce apoptosis in at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, or at leastabout 55% of cells. In some particular embodiments, the CD37-bindingagents are capable of inducing apoptosis in Ramos cells and/or Rajicells.

In some embodiments, the CD37-binding agents are capable of depletingB-cells. In some embodiments, the B-cells are autoreactive B-cells. Insome embodiments, the B-cells are not cancer cells. In some embodiments,the B-cells are not tumor cells. In some embodiments, the B-cells arenot cancerous cells. In some embodiments, the B-cells overexpress CD37.In some embodiments, the B-cells do not overexpress CD37.

Treatment of cells with CD37-binding agents can result in depletion ofat least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 55%,at least about 60%, at least about 65%, at least about 70%, or leastabout 75% of B-cells.

In some embodiments, the CD37-binding agents do not deplete T-cellsunder the same conditions in which B-cells are depleted. For example,treatment of cells with CD37-binding agents can result in depletion ofless than about 20%, less than about 15%, less than about 10%, or lessthan about 5% of T-cells. In certain embodiments, the CD37-bindingagents deplete at least about 25% of B-cells and deplete less than about10% of T-cells. In certain embodiments, the CD37-binding agents depleteat least about 30% of B-cells and deplete less than about 5% of T-cells.

In some embodiments, the CD37-binding agents do not deplete monocytesunder the same conditions in which B-cells are depleted. For example,treatment of cells with CD37-binding agents can result in depletion ofless than about 20%, less than about 15%, less than about 10%, or lessthan about 5% of monocytes. In certain embodiments, the CD37-bindingagents deplete at least about 25% of B-cells and deplete less than about10% of monocytes. In certain embodiments, the CD37-binding agentsdeplete at least about 30% of B-cells and deplete less than about 5% ofmonocytes.

In certain embodiments, immunoconjugates or other agents thatspecifically bind human CD37 trigger cell death via a cytotoxic agent.For example, in certain embodiments, an antibody to human CD37 isconjugated to a maytansinoid that is activated in cells expressing theCD37 by protein internalization. In certain alternative embodiments, theagent or antibody is not conjugated to a maytansinoid or other cytotoxicmolecule.

The CD37-binding agents include CD37 antibodies such as CD37-3, CD37-12,CD37-38, CD37-50, CD37-51, CD37-56 and CD37-57 and fragments, variantsand derivatives thereof. The CD37-binding agents also includeCD37-binding agents that specifically bind to the same CD37 epitope asan antibody selected from the group consisting of CD37-3, CD37-12,CD37-38, CD37-50, CD37-51, CD37-56 and CD37-57. The CD37-binding agentsalso include CD37-binding agents that competitively inhibit an antibodyselected from the group consisting of CD37-3, CD37-12, CD37-38, CD37-50,CD37-51, CD37-56 and CD37-57.

In some particular embodiments, CD37-binding agents can be characterizedby their ability to bind chimeric CD37 polypeptides, includingmurine/human and macaca/human chimeric polypeptides described in U.S.Published Application No. 2011/0256153, which is herein incorporated byreference in its entirety, and provided in the table below.

Chimeric Poly- peptide Sequence hCD37-MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKV M1LAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRVRLERRVQELVLRTIQSYRTNPDETAAEESWDYVQFQLRCCGWHYPQDWFQVLILRGNGSEAHRVPCSCYNLSATNDSTILDKVILPQLSRLGHLARSRHSADICAVPAESHIYREGCAQGLQKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARYR  (SEQ ID NO: 184)muCD37- ISTQRVRLERRVQELVLRTIQSYRTNPDETAAEESWDYAQFQLRCCGWQSPRDWNK R176AQMLKANESEEPRVPCSCYNSTATNDSTVFDKLFFSQLSRLGPRAKLRQTADICALPAKAHIYREGCAQSLQ (SEQ ID NO: 185) hCD37-MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKV M45LAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRAQLERSLRDVVEKTIQKYGTNPEETAAEESWDYVQFQLRCCGWHYPQDWFQVLILRGNGSEAHRVPCSCYNLSATNDSTILDKVILPQLSRLGPRAKLRQTADICALPAKAHIYREGCAQSLQKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARYR  (SEQ ID NO: 186)hCD37m MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKV ECD-LAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRVRLERRV H45QELVLRTIQSYRTNPDETAAEESWDYAQFQLRCCGWQSPRDWNKAQMLKANESEEPRVPCSCYNSTATNDSTVFDKLFFSQLSRLGHLARSRHSADICAVPAESHIYREGCAQGLQKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARYR  (SEQ ID NO: 187)hCD37m MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKVECD-H5 LAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRVRLERRVQELVLRTIQSYRTNPDETAAEESWDYAQFQLRCCGWQSPRDWNKAQMLKANESEEPRVPCSCYNSTATNDSTVFDKLFFSQLSRLGPRAKLRQTADICAVPAESHIYREGCAQGLQKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARYR  (SEQ ID NO: 188)hCD37m MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKVECD-H4 LAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRVRLERRVQELVLRTIQSYRTNPDETAAEESWDYAQFQLRCCGWQSPRDWNKAQMLKANESEEPRVPCSCYNSTATNDSTVFDKLFFSQLSRLGHLARSRHSADICALPAKAHIYREGCAQSLQKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARYR  (SEQ ID NO: 189)hCD37- MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKV Mac4LAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRAQLERSLRDVVEKTIQKYGTNPEETAAEESWDYVQFQLRCCGWHYPQDWFQVLILRGNGSEAHRVPCSCYNLSATNDSTILDKVILPQLSRLGQLARSRHSTDICAVPAESHIYREGCAQGLQKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARYR  (SEQ ID NO: 190) hCD37-MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKV Mac45LAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRAQLERSLRDVVEKTIQKYGTNPEETAAEESWDYVQFQLRCCGWHYPQDWFQVLILRGNGSEAHRVPCSCYNLSATNDSTILDKVILPQLSRLGQLARSRHSTDICAVPANSHIYREGCARSLQKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARY (SEQ ID NO: 191) hCD37-MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKV Mac5LAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRAQLERSLRDVVEKTIQKYGTNPEETAAEESWDYVQFQLRCCGWHYPQDWFQVLILRGNGSEAHRVPCSCYNLSATNDSTILDKVILPQLSRLGHLARSRHSADICAVPANSHIYREGCARSLQKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARYR  (SEQ ID NO: 192)

In some particular embodiments, the binding of the CD37-binding agentsto CD37 does not require human CD37 amino acids 109-138. Thus, someCD37-binding agents bind to a polypeptide comprising the amino acidsequence of SEQ ID NO:184. In other embodiments, the binding of theCD37-binding agents to CD37 is disrupted by mutation of human CD37 aminoacids 202-243. Thus, some CD37-binding agents do not bind to apolypeptide comprising the amino acid sequence of SEQ ID NO:185.

In some embodiments, the CD37-binding agents bind to a polypeptide ofSEQ ID NO:184 and to a polypeptide of SEQ ID NO:186, but do not bind toa polypeptide of SEQ ID NO:185.

In some embodiments, the CD37-binding agents bind to a polypeptide ofSEQ ID NO:187. In some embodiments, the CD37-binding agents bind to apolypeptide of SEQ ID NO:187 and a polypeptide of SEQ ID NO:188. In someembodiments, the CD37-binding agents bind to a polypeptide of SEQ IDNO:187 and a polypeptide of SEQ ID NO:189.

In some embodiments, the CD37-binding agent binds to a polypeptide ofSEQ ID NO:190, but does not bind to a polypeptide of SEQ ID NO:191. Insome embodiments, the CD37-binding agent binds to a polypeptide of SEQID NO:192, but does not bind to a polypeptide of SEQ ID NO:191.

CD37 peptide fragments to which certain CD37-binding agents bind toinclude, but are not limited to, CD37 fragments comprising, consistingessentially of, or consisting of amino acids 200-243 of SEQ ID NO: 1,amino acids 202-220 or SEQ ID NO:1, or amino acids 221-243 of SEQ IDNO:1. In some embodiments, the CD37-binding agent is specifically bindsto a human CD37 epitope comprising amino acids 202-243 of SEQ ID NO:1.In some embodiments, the binding of the CD37-binding agent to CD37requires amino acids 202-243 of SEQ ID NO:1. In some embodiments, thebinding of the CD37-binding agent to CD37 requires amino acids 200-220of SEQ ID NO: 1. In some embodiments, the binding of the CD37-bindingagent to CD37 requires amino acids 221-243 of SEQ ID NO:1.

Examples of CD37-binding agents with the aforementioned bindingproperties are described in U.S. Published Application No. 2011/0256153,which is herein incorporated by reference in its entirety.

The CD37-binding agents also include CD37-binding agents that comprisethe heavy and light chain CDR sequences of CD37-3, CD37-12, CD37-38,CD37-50, CD37-51, CD37-56 or CD37-57. The heavy and light chain CDRs ofCD37-38, CD37-50, CD37-51, CD37-56 and CD37-57 contain relatedsequences. Therefore, the CD37-binding agents can also comprise heavyand light chain CDR sequences that comprise a consensus sequenceobtained by the alignment of CD37-38, CD37-50, CD37-51, CD37-56 andCD37-57. The CDR sequences of CD37-3, CD37-12, CD37-38, CD37-50,CD37-51, CD37-56 and CD37-57, as well as the consensus sequence ofCD37-38, CD37-50, CD37-51, CD37-56 and CD37-57 are described in Tables 1and 2 below.

TABLE 1  Variable heavy chain CDR amino acid sequences Antibody VH-CDR1VH-CDR2 VH-CDR3 CD37-3 TSGVS (SEQ ID VIWGDGSTN (SEQ IDGGYSLAH (SEQ ID NO: 6) NO: 4) NO: 5) CD37-12 KYGMN (SEQ IDWINTNTGESR (SEQ ID GTVVAD (SEQ ID NO: 9) NO: 7) NO: 8) CD37-38SGFGWH (SEQ ID YILYSGGTD (SEQ ID GYYGYGAWFVY (SEQ ID NO: 10) NO: 11)NO: 12) CD37-50 SGFAWH (SEQ ID YILYSGSTV (SEQ ID GYYGYGAWFAY (SEQ IDNO: 13) NO: 14) NO: 15) CD37-51 SGFAWH (SEQ ID YIHYSGSTN (SEQ IDGYYGFGAWFVY (SEQ ID NO: 16) NO: 17) NO: 18) CD37-56 SGFAWH (SEQ IDYIHYSGGTN (SEQ ID GYYGFGAWFAY (SEQ ID NO: 19) NO: 20) NO: 21) CD37-57SGFAWH (SEQ ID YILYSGSTV (SEQ ID GYYGYGAWFAY (SEQ ID NO: 22) NO: 23)NO: 24) CONSENSUS SGF[A or G]WH YI[L or H]YSG GYYG[Y or F]GAWF[V or(SEQ ID NO: 25) [G or SN] (SEQ ID A]Y (SEQ ID NO: 27) NO: 26) 252-3SYGMS (SEQ ID TISSGGSYTYSPDSVKG HSYYDTSVDY NO: 171) (SEQ ID NO: 172)(SEQ ID NO: 173) 252-3 SYGMS (SEQ ID TISSGGSYTY (SEQ IDHSYYDTSVDY (SEQ ID NO: 171) NO: 181) NO: 173)

TABLE 2  Variable light chain CDR amino acid sequences Antibody VL-CDR1VL-CDR2 VL-CDR3 CD37-3 RASENIRSNLA (SEQ VATNLAD  QHYWGTTWT  (SEQ ID(SEQ ID ID NO: 28) NO: 29) NO: 30) CD37-12 RASQSVSTSSYSYLY YASNLAS QHSWEIPYT  (SEQ ID (SEQ ID (SEQ ID NO: 31) NO: 32) NO: 33) CD37-38SASSSVTYMH (SEQ DTSKLAS  QQWISNPPT  (SEQ ID (SEQ ID ID NO: 34) NO: 35)NO: 36) CD37-50 SATSSVTYMH (SEQ DTSKLPY  QQWSDNPPT  (SEQ ID (SEQ IDID NO: 37) NO: 38) NO: 39) Humanized DTSNLPY  (SEQ ID NO: 40) CD37-51SATSSVTYMH (SEQ DTSKLAS  QQWSSNPPT  (SEQ ID (SEQ ID ID NO: 41) NO: 42)NO: 43) CD37-56 SASSSVTYMH (SEQ DTSKLAS  QQWISDPPT  (SEQ ID (SEQ IDID NO: 44) NO: 45) NO: 46) Humanized DTSNLAS (SEQ ID NO: 47) CD37-57SATSSVTYMH (SEQ DTSKLAS  QQWSDNPPT  (SEQ ID (SEQ ID ID NO: 48) NO: 49)NO: 50) Humanized DTSNLAS (SEQ ID  NO: 51) CONSENSUS SA[T or S]DTS[K or N] QQW[I or S] SSVTYMH L[A or P] [S or D] (SEQ ID NO: 52)[S or Y] [N or D]PPT  (SEQ ID  (SEQ ID  NO: 53) NO: 54) 252-3RASQDISNYLN (SEQ YTSKLHS  QQGNALPWT  ID NO: 174) (SEQ ID (SEQ IDNO: 175) NO: 176)

The CD37 binding molecules can be antibodies or antigen bindingfragments that specifically bind to CD37 that comprise the CDRs ofCD37-3, CD37-12, CD37-50, CD37-51, CD37-56, or CD37-57 with up to four(i.e., 0, 1, 2, 3, or 4) conservative amino acid substitutions per CDR.

The CD37 binding molecules can comprise one of the individual variablelight chains or variable heavy chains described herein. Antibodies andpolypeptides can also comprise both a variable light chain and avariable heavy chain. The variable light chain and variable heavy chainsequences of murine, chimeric, and humanized CD37-3, CD37-12, CD37-50,CD37-51, CD37-56, and CD37-57 antibodies are provided in Tables 3 and 4below.

TABLE 3  Variable heavy chain amino acid sequences AntibodyVH Amino Acid Sequence (SEQ ID NO) muCD37-3QVQVKESGPGLVAPSQSLSITCTVSGFSLTTSGVSWVRQPPGKGLEWLGVIWGDGSTNYHSALKSRLSIKKDHSKSQVFLKLNSLQTDDTATYYCAKGGYSLAHWGQGTLVTVSA (SEQ ID NO: 55) chCD37-3QVQVKESGPGLVAPSQSLSITCTVSGFSLTTSGVSWVRQPPGKGLEWLGVIWGDGSTNYHSALKSRLSIKKDHSKSQVFLKLNSLQTDDTATYYCAKGGYSLAHWGQGTLVTVSA (SEQ ID NO: 56) huCD37-3v1.0QVQVQESGPGLVAPSQTLSITCTVSGFSLTTSGVSWVRQPPGKGLEWLGVIWGDGSTNYHPSLKSRLSIKKDHSKSQVFLKLNSLTAADTATYYCAKGGYSLAHWGQGTLVTVSS (SEQ ID NO: 57) huCD37-3v1.1QVQVQESGPGLVAPSQTLSITCTVSGFSLTTSGVSWVRQPPGKGLEWLGVIWGDGSTNYHSSLKSRLSIKKDHSKSQVFLKLNSLTAADTATYYCAKGGYSLAHWGQGTLVTVSS (SEQ ID NO: 58) muCD37-12QIQLVQSGPELKKPGETVKISCKASGYTFTKYGMNWVKQAQGKGLKWMGWINTNTGESRNAEEFKGRFAFSLETSASTAYLQINNLKYEDTATYFCGRGTVVADWGQGTTLTVSS (SEQ ID NO: 59) chCD37-12QIQLVQSGPELKKPGETVKISCKASGYTFTKYGMNWVKQAQGKGLKWMGWINTNTGESRNAEEFKGRFAFSLETSASTAYLQINNLKYEDTATYFCGRGTVVADWGQGTTLTVSS (SEQ ID NO: 60) muCD37-38DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGFGWHWIRQFPGNKLEWMAYILYSGGTDYNPSLKSRISITRDTSKNQFFLRLSSVTTEDTATYYCARGYYGYGAWFVYWGQGTLVTVSA (SEQ ID NO: 61) chCD37-38QVQLQESGPDLVKPSQSLSLTCTVTGYSITSGFGWHWIRQFPGNKLEWMAYILYSGGTDYNPSLKSRISITRDTSKNQFFLRLSSVTTEDTATYYCARGYYGYGAWFVYWGQGTLVTVSA (SEQ ID NO: 62) huCD37-38QVQLQESGPGLVKPSQSLSLTCTVSGYSITSGFGWHWIRQFPGKGLEWMAYILYSGGTDYNPSLKSRISITRDTSKNQFFLRLSSVTAADTATYYCARGYYGYGAWFVYWGQGTLVTVSS (SEQ ID NO: 63) muCD37-50DVQLQESGPDLLKPSQSLSLTCTVTGYSITSGFAWHWIRQFPGNKLEWMGYILYSGSTVYSPSLKSRISITRDTSKNHFFLQLNSVTTEDTATYYCARGYYGYGAWFAYWGQGTLVTVSA (SEQ ID NO: 64) huCD37-50QVQLQESGPGLLKPSQSLSLTCTVSGYSITSGFAWHWIRQHPGNKLEWMGYILYSGSTVYSPSLKSRISITRDTSKNHFFLQLNSVTAADTATYYCARGYYGYGAWFAYWGQGTLVTVSA (SEQ ID NO: 65) muCD37-51DVQLQESGPDLLKPSQSLSLTCTVTGYSISSGFAWHWIRQFPGNKLEWMGYIHYSGSTNYSPSLKSRISITRDSSKNQFFLQLNSVTTEDTATYYCARGYYGFGAWFVYWGQGTLVTVSA (SEQ ID NO: 66) huCD37-51EVQLVESGPEVLKPGESLSLTCTVSGYSISSGFAWHWIRQFPGKGLEWMGYIHYSGSTNYSPSLQGRISITRDSSINQFFLQLNSVTASDTATYYCARGYYGFGAWFVYWGQGTLVTVSA (SEQ ID NO: 67) muCD37-56DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGFAWHWIRQFPGNKLEWMGYIHYSGGTNYNPSLKSRVSITRDTSKNQFFLQLNSVTTEDTATYYCARGYYGFGAWFAYWGQGTLVPVSA (SEQ ID NO: 68) huCD37-56QVQLQESGPGLVKPSQSLSLTCTVSGYSITSGFAWHWIRQFPGKGLEWMGYIHYSGGTNYNPSLKSRVSITRDTSKNQFFLQLNSVTAADTATYYCARGYYGFGAWFAYWGQGTLVPVSA (SEQ ID NO: 69) muCD37-57DVQLQESGPDLLKPSQSLSLTCTVTGYSITSGFAWHWIRQFPGNKLEWMGYILYSGSTVYSPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARGYYGYGAWFAYWGQGTLVTVSA (SEQ ID NO: 70) huCD37-57QVQLQESGPGLLKPSQSLSLTCTVSGYSITSGFAWHWIRQFPGKGLEWMGYILYSGSTVYSPSLKSRISITRDTSKNQFFLQLNSVTAADTATYYCARGYYGYGAWFAYWGQGTLVTVSA (SEQ ID NO: 71) 252-3EVQVVESGGDLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPDKRLEWVATISSGGSYTYSPDSVKGRFTISRDNAKKTLYLQMSSLKSEDTAMYYCARHSYYDTSVDYWGQGTSVTVSS (SEQ ID NO: 177)

TABLE 4  Variable light chain amino acid sequences AntibodyVL Amino Acid Sequence (SEQ ID NO) muCD37-3DIQMTQSPASLSVSVGETVTITCRASENIRSNLAWYQQKQGKSPQLLVNVATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHYWGTTWTFGGGTKLEIKR (SEQ ID NO: 72) chCD37-3DIQMTQSPASLSVSVGETVTITCRASENIRSNLAWYQQKQGKSPQLLVNVATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHYWGTTWTFGGGTKLEIKR (SEQ ID NO: 73) huCD37-3DIQMTQSPSSLSVSVGERVTITCRASENIRSNLAWYQQKPGKSPKLLVNVAT (1.0 and 1.1)NLADGVPSRFSGSGSGTDYSLKINSLQPEDFGTYYCQHYWGTTWTFGQGTKLEIKR (SEQ ID NO: 74) muCD37-12DIVLTQSPASLAVSLGQRATISCRASQSVSTSSYSYLYWFQQKPGQPPKLLIKYASNLASGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPYTFGGGTKLEIKR (SEQ ID NO: 75) chCD37-12DIVLTQSPASLAVSLGQRATISCRASQSVSTSSYSYLYWFQQKPGQPPKLLIKYASNLASGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPYTFGGGTKLEIKR (SEQ ID NO: 76) muCD37-38QIVLTQSPAIMSASPGEKVTMTCSASSSVTYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGGGSGTSYSLTISSMEAEDAATYYCQQWISNPPTFGGGTKLEIKR (SEQ ID NO: 77) chCD37-38QIVLTQSPAIMSASPGEKVTMTCSASSSVTYMHWYQQKSGTSPKRWIYDTSKLASGVPARF SGGGSGTSYSLTISSMEAEDAATYYCQQWISNPPTFGGGTKLEIKR (SEQ ID NO: 78) huCD37-38DIVLTQSPASMSASPGERVTMTCSASSSVTYMHWYQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWISNPPTFGGGTKLEIKR (SEQ ID NO: 79) muCD37-50QIVLTQSPAIMSASPGEKVTMTCSATSSVTYMHWYQQKSGTSPKRWIYDTSKLPYGVPGRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSDNPPTFGSGTKLEIKR (SEQ ID NO: 80) huCD37-50EIVLTQSPATMSASPGERVTMTCSATSSVTYMHWYQQKPGQSPKRWIYDTSNLPYGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSDNPPTFGQGTKLEIKR (SEQ ID NO: 81) muCD37-51QIVLTQSPAIMSASPGEKVTMTCSATSSVTYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISNMEAEDAATYYCQQWSSNPPTFGSGTKLEIKR (SEQ ID NO: 82) huCD37-51EIVLTQSPATMSASPGERVTMTCSATSSVTYMHWYQQKPGQSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPTFGQGTKLEIKR (SEQ ID NO: 83) muCD37-56QIVLTQSPAFMSASPGDKVTMTCSASSSVTYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGGGSGTSYSLTISTMEAEDAATYYCQQWISDPPTFGGGTKLEIKR (SEQ ID NO: 84) huCD37-56DIVLTQSPAFMSASPGEKVTMTCSASSSVTYMHWYQQKPDQSPKRWIYDTSNLASGVPSRFSGGGSGTDYSLTISSMEAEDAATYYCQQWISDPPTFGQGTKLEIKR (SEQ ID NO: 85) muCD37-57QIVLTQSPAIMSASPGEKVTMTCSATSSVTYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSDNPPTFGSGTKLEIKR (SEQ ID NO: 86) huCD37-57EIVLTQSPATMSASPGERVTMTCSATSSVTYMHWYQQKPGQSPRRWIYDTSNLASGVPARF SGSGSGTSYSLTISSMEAEDAATYYCQQWSDNPPTFGQGTKLEIKR (SEQ ID NO: 87) 252-3DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSKLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNALPWTFGGGTKLELKR (SEQ ID NO: 178)

Also provided are polypeptides that comprise: (a) a polypeptide havingat least about 90% sequence identity to SEQ ID NOs:55-71 or 177; and/or(b) a polypeptide having at least about 90% sequence identity to SEQ IDNOs:72-87 or 178. In certain embodiments, the polypeptide comprises apolypeptide having at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, or at least about 99% sequence identityto SEQ ID NOs:55-87, 177, or 178. Thus, in certain embodiments, thepolypeptide comprises (a) a polypeptide having at least about 95%sequence identity to SEQ ID NOs:55-71 or 177, and/or (b) a polypeptidehaving at least about 95% sequence identity to SEQ ID NOs:72-87 or 178.In certain embodiments, the polypeptide comprises (a) a polypeptidehaving the amino acid sequence of SEQ ID NOs:55-71 or 177; and/or (b) apolypeptide having the amino acid sequence of SEQ ID NOs:72-87 or 178.In certain embodiments, the polypeptide is an antibody and/or thepolypeptide specifically binds CD37. In certain embodiments, thepolypeptide is a murine, chimeric, or humanized antibody thatspecifically binds CD37. In certain embodiments, the polypeptide havinga certain percentage of sequence identity to SEQ ID NOs:55-87, 177, or178 differs from SEQ ID NOs:55-87 by conservative amino acidsubstitutions only.

Polypeptides can comprise one of the individual light chains or heavychains described herein. Antibodies and polypeptides can also compriseboth a light chain and a heavy chain. The light chain and variable chainsequences of murine, chimeric, and humanized CD37-3, CD37-12, CD37-50,CD37-51, CD37-56, and CD37-57 antibodies are provided in Tables 5 and 6below.

TABLE 5  Full-length heavy chain amino acid sequences AntibodyFull-Length Heavy Chain Amino Acid Sequence (SEQ ID NO) muCD37-3QVQVKESGPGLVAPSQSLSITCTVSGFSLTTSGVSWVRQPPGKGLEWLGVIWGDGSTNYHSALKSRLSIKKDHSKSQVFLKLNSLQTDDTATYYCAKGGYSLAHWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHEITTKSFSRTPGK (SEQ ID NO: 88) chCD37-3QVQVKESGPGLVAPSQSLSITCTVSGFSLTTSGVSWVRQPPGKGLEWLGVIWGDGSTNYHSALKSRLSIKKDHSKSQVFLKLNSLQTDDTATYYCAKGGYSLAHWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 89) huCD37-3v1.0QVQVQESGPGLVAPSQTLSITCTVSGFSLTTSGVSWVRQPPGKGLEWLGVIWGDGSTNYHPSLKSRLSIKKDHSKSQVFLKLNSLTAADTATYYCAKGGYSLAHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 90) huCD37-3v1.1QVQVQESGPGLVAPSQTLSITCTVSGFSLTTSGVSWVRQPPGKGLEWLGVIWGDGSTNYHSSLKSRLSIKKDHSKSQVFLKLNSLTAADTATYYCAKGGYSLAHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 91) muCD37-12QIQLVQSGPELKKPGETVKISCKASGYTFTKYGMNWVKQAQGKGLKWMGWINTNTGESRNAEEFKGRFAFSLETSASTAYLQINNLKYEDTATYFCGRGTVVADWGQGTTLTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHEITTKSFSRTPGK (SEQ ID NO: 92) chCD37-12QIQLVQSGPELKKPGETVKISCKASGYTFTKYGMNWVKQAQGKGLKWMGWINTNTGESRNAEEFKGRFAFSLETSASTAYLQINNLKYEDTATYFCGRGTVVADWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 93) muCD37-38DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGFGWHWIRQFPGNKLEWMAYILYSGGTDYNPSLKSRISITRDTSKNQFFLRLSSVTTEDTATYYCARGYYGYGAWFVYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLESDLYTLSSSVTVPSSMRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHEITEKSLSHSPGK (SEQ ID NO: 94) chCD37-38QVQLQESGPDLVKPSQSLSLTCTVTGYSITSGFGWHWIRQFPGNKLEWMAYILYSGGTDYNPSLKSRISITRDTSKNQFFLRLSSVTTEDTATYYCARGYYGYGAWFVYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 95) huCD37-38QVQLQESGPGLVKPSQSLSLTCTVSGYSITSGFGWHWIRQFPGKGLEWMAYILYSGGTDYNPSLKSRISITRDTSKNQFFLRLSSVTAADTATYYCARGYYGYGAWFVYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 96) mu CD37-50DVQLQESGPDLLKPSQSLSLTCTVTGYSITSGFAWHWIRQFPGNKLEWMGYILYSGSTVYSPSLKSRISITRDTSKNHFFLQLNSVTTEDTATYYCARGYYGYGAWFAYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHEITTKSFSRTPGK (SEQ ID NO: 97) huCD37-50QVQLQESGPGLLKPSQSLSLTCTVSGYSITSGFAWHWIRQHPGNKLEWMGYILYSGSTVYSPSLKSRISITRDTSKNHFFLQLNSVTAADTATYYCARGYYGYGAWFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 98) mu CD37-51DVQLQESGPDLLKPSQSLSLTCTVTGYSISSGFAWHWIRQFPGNKLEWMGYIHYSGSTNYSPSLKSRISITRDSSKNQFFLQLNSVTTEDTATYYCARGYYGFGAWFVYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHEITTKSFSRTPGK (SEQ ID NO: 99) huCD37-51EVQLVESGPEVLKPGESLSLTCTVSGYSISSGFAWHWIRQFPGKGLEWMGYIHYSGSTNYSPSLQGRISITRDSSINQFFLQLNSVTASDTATYYCARGYYGFGAWFVYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 100) muCD37-56DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGFAWHWIRQFPGNKLEWMGYIHYSGGTNYNPSLKSRVSITRDTSKNQFFLQLNSVTTEDTATYYCARGYYGFGAWFAYWGQGTLVPVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLESDLYTLSSSVTVPSSMRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHEITEKSLSHSPGK (SEQ ID NO: 101) huCD37-56QVQLQESGPGLVKPSQSLSLTCTVSGYSITSGFAWHWIRQFPGKGLEWMGYIHYSGGTNYNPSLKSRVSITRDTSKNQFFLQLNSVTAADTATYYCARGYYGFGAWFAYWGQGTLVPVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 102) muCD37-57DVQLQESGPDLLKPSQSLSLTCTVTGYSITSGFAWHWIRQFPGNKLEWMGYILYSGSTVYSPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARGYYGYGAWFAYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISL SPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHEITTKSFSRTPGK (SEQ ID NO: 103) huCD37-57QVQLQESGPGLLKPSQSLSLTCTVSGYSITSGFAWHWIRQFPGKGLEWMGYILYSGSTVYSPSLKSRISITRDTSKNQFFLQLNSVTAADTATYYCARGYYGYGAWFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 104) 252-3EVQVVESGGDLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPDKRLEWVATISSGGSYTYSPDSVKGRFTISRDNAKKTLYLQMSSLKSEDTAMYYCARHSYYDTSVDYWGQGTSVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHEITTKSFSRTPGK (SEQ ID NO: 179)

TABLE 6  Full-length light chain amino acid sequences AntibodyFull-length Light Chain Amino Acid Sequence (SEQ ID NO) muCD37-3DIQMTQSPASLSVSVGETVTITCRASENIRSNLAWYQQKQGKSPQLLVNVATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHYWGTTWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 105) chCD37-3DIQMTQSPASLSVSVGETVTITCRASENIRSNLAWYQQKQGKSPQLLVNVATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHYWGTTWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 106) huCD37-3DIQMTQSPSSLSVSVGERVTITCRASENIRSNLAWYQQKPGKSPKLLVNVAT (1.0 and 1.1)NLADGVPSRFSGSGSGTDYSLKINSLQPEDFGTYYCQHYWGTTWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 107) muCD37-12DIVLTQSPASLAVSLGQRATISCRASQSVSTSSYSYLYWFQQKPGQPPKLLIKYASNLASGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 108) chCD37-12DIVLTQSPASLAVSLGQRATISCRASQSVSTSSYSYLYWFQQKPGQPPKLLIKYASNLASGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 109) muCD37-38QIVLTQSPAIMSASPGEKVTMTCSASSSVTYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGGGSGTSYSLTISSMEAEDAATYYCQQWISNPPTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 110) chCD37-38QIVLTQSPAIMSASPGEKVTMTCSASSSVTYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGGGSGTSYSLTISSMEAEDAATYYCQQWISNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 111) huCD37-38DIVLTQSPASMSASPGERVTMTCSASSSVTYMHWYQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWISNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 112) muCD37-50QIVLTQSPAIMSASPGEKVTMTCSATSSVTYMHWYQQKSGTSPKRWIYDTSKLPYGVPGRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSDNPPTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 113) huCD37-50EIVLTQSPATMSASPGERVTMTCSATSSVTYMHWYQQKPGQSPKRWIYDTSNLPYGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSDNPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 114) muCD37-51QIVLTQSPAIMSASPGEKVTMTCSATSSVTYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISNMEAEDAATYYCQQWSSNPPTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 115) huCD37-51EIVLTQSPATMSASPGERVTMTCSATSSVTYMHWYQQKPGQSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 116) muCD37-56QIVLTQSPAFMSASPGDKVTMTCSASSSVTYMEIWYQQKSGTSPKRWIYDTSKLASGVPARFSGGGSGTSYSLTISTMEAEDAATYYCQQWISDPPTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 117) huCD37-56DIVLTQSPAFMSASPGEKVTMTCSASSSVTYMHWYQQKPDQSPKRWIYDTSNLASGVPSRFSGGGSGTDYSLTISSMEAEDAATYYCQQWISDPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 118) muCD37-57QIVLTQSPAIMSASPGEKVTMTCSATSSVTYMHWYQQKSGTSPKRWIYDTS KLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSDNPPTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 119) huCD37-57EIVLTQSPATMSASPGERVTMTCSATSSVTYMHWYQQKPGQSPRRWIYDTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSDNPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 120) 252-3DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSKLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNALPWTFGGGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 180)

Also provided are polypeptides that comprise: (a) a polypeptide havingat least about 90% sequence identity to SEQ ID NOs:88-104 or 179; and/or(b) a polypeptide having at least about 90% sequence identity to SEQ IDNOs:105-120 or 180. In certain embodiments, the polypeptide comprises apolypeptide having at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, or at least about 99% sequence identityto SEQ ID NOs:88-120, 179, or 180. Thus, in certain embodiments, thepolypeptide comprises (a) a polypeptide having at least about 95%sequence identity to SEQ ID NOs:88-104 or 179, and/or (b) a polypeptidehaving at least about 95% sequence identity to SEQ ID NOs:105-120 or180. In certain embodiments, the polypeptide comprises (a) a polypeptidehaving the amino acid sequence of SEQ ID NOs:88-104 or 179; and/or (b) apolypeptide having the amino acid sequence of SEQ ID NOs:105-120 or 180.In certain embodiments, the polypeptide is an antibody and/or thepolypeptide specifically binds CD37. In certain embodiments, thepolypeptide is a murine, chimeric, or humanized antibody thatspecifically binds CD37. In certain embodiments, the polypeptide havinga certain percentage of sequence identity to SEQ ID NOs:88-120, 179, or180 differs from SEQ ID NOs:88-120, 179, or 180 by conservative aminoacid substitutions only.

In certain embodiments, the CD37 antibody can be the antibody producedfrom a hybridoma selected from the group consisting of consisting ofATCC Deposit Designation PTA-10664, deposited with the ATCC on Feb. 18,2010, ATCC Deposit Designation PTA-10665, deposited with the ATCC onFeb. 18, 2010, ATCC Deposit Designation PTA-10666, deposited with theATCC on Feb. 18, 2010, ATCC Deposit Designation PTA-10667 deposited withthe ATCC on Feb. 18, 2010, ATCC Deposit Designation PTA-10668, depositedwith the ATCC on Feb. 18, 2010, ATCC Deposit Designation PTA-10669,deposited with the ATCC on Feb. 18, 2010, and ATCC Deposit DesignationPTA-10670, deposited with the ATCC on Feb. 18, 2010 (American TypeCulture Collection (ATCC) at 10801 University Boulevard, Manassas, Va.20110). In certain embodiments, the antibody comprises the VH-CDRs andthe VL-CDRS of the antibody produced from a hydridoma selected from thegroup consisting of PTA-10665, PTA-10666, PTA-10667, PTA-10668,PTA-10669, and PTA-10670.

In certain embodiments, the CD37 antibody can comprise a light chainencoded by the recombinant plasmid DNA phuCD37-3LC (ATCC DepositDesignation PTA-10722, deposited with the ATCC on Mar. 18, 2010). Incertain embodiments, the CD37 antibody can comprise a heavy chainencoded by the recombinant plasmid DNA phuCD37-3HCv.1.0 (ATCC DepositDesignation PTA-10723, deposited with the ATCC on Mar. 18, 2010). Incertain embodiments, the CD37 antibody can comprise a light chainencoded by the recombinant plasmid DNA phuCD37-3LC (PTA-10722) and aheavy chain encoded by the recombinant plasmid DNA phuCD37-3HCv.1.0(PTA-10723). In certain embodiments, the CD37 antibody can comprise theVL-CDRs encoded by the recombinant plasmid DNA phuCD37-3LC (PTA-10722)and the VH-CDRs encoded by the recombinant plasmid DNA phuCD37-3HCv.1.0(PTA-10723).

Monoclonal antibodies can be prepared using hybridoma methods, such asthose described by Kohler and Milstein (1975) Nature 256:495. Using thehybridoma method, a mouse, hamster, or other appropriate host animal, isimmunized as described above to elicit the production by lymphocytes ofantibodies that will specifically bind to an immunizing antigen.Lymphocytes can also be immunized in vitro. Following immunization, thelymphocytes are isolated and fused with a suitable myeloma cell lineusing, for example, polyethylene glycol, to form hybridoma cells thatcan then be selected away from unfused lymphocytes and myeloma cells.Hybridomas that produce monoclonal antibodies directed specificallyagainst a chosen antigen as determined by immunoprecipitation,immunoblotting, or by an in vitro binding assay (e.g. radioimmunoassay(RIA); enzyme-linked immunosorbent assay (ELISA)) can then be propagatedeither in vitro culture using standard methods (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, 1986) or in vivo asascites tumors in an animal. The monoclonal antibodies can then bepurified from the culture medium or ascites fluid as described forpolyclonal antibodies above.

Alternatively monoclonal antibodies can also be made using recombinantDNA methods as described in U.S. Pat. No. 4,816,567. The polynucleotidesencoding a monoclonal antibody are isolated from mature B-cells orhybridoma cell, such as by RT-PCR using oligonucleotide primers thatspecifically amplify the genes encoding the heavy and light chains ofthe antibody, and their sequence is determined using conventionalprocedures. The isolated polynucleotides encoding the heavy and lightchains are then cloned into suitable expression vectors, which whentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, monoclonal antibodies aregenerated by the host cells. Also, recombinant monoclonal antibodies orfragments thereof of the desired species can be isolated from phagedisplay libraries expressing CDRs of the desired species as described(McCafferty et al., 1990, Nature, 348:552-554; Clackson et al., 1991,Nature, 352:624-628; and Marks et al., 1991, J. Mol. Biol.,222:581-597).

The polynucleotide(s) encoding a monoclonal antibody can further bemodified in a number of different manners using recombinant DNAtechnology to generate alternative antibodies. In some embodiments, theconstant domains of the light and heavy chains of, for example, a mousemonoclonal antibody can be substituted 1) for those regions of, forexample, a human antibody to generate a chimeric antibody or 2) for anon-immunoglobulin polypeptide to generate a fusion antibody. In someembodiments, the constant regions are truncated or removed to generatethe desired antibody fragment of a monoclonal antibody. Site-directed orhigh-density mutagenesis of the variable region can be used to optimizespecificity, affinity, etc. of a monoclonal antibody.

In some embodiments, the monoclonal antibody against the human CD37 is ahumanized antibody. In certain embodiments, such antibodies are usedtherapeutically to reduce antigenicity and HAMA (human anti-mouseantibody) responses when administered to a human subject. Humanizedantibodies can be produced using various techniques known in the art. Incertain alternative embodiments, the antibody to CD37 is a humanantibody.

Human antibodies can be directly prepared using various techniques knownin the art. Immortalized human B lymphocytes immunized in vitro orisolated from an immunized individual that produce an antibody directedagainst a target antigen can be generated (See, e.g., Cole et al.,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985);Boemer et al., 1991, J. Immunol., 147 (1):86-95; and U.S. Pat. No.5,750,373). Also, the human antibody can be selected from a phagelibrary, where that phage library expresses human antibodies, asdescribed, for example, in Vaughan et al., 1996, Nat. Biotech.,14:309-314, Sheets et al., 1998, Proc. Nat'l. Acad. Sci., 95:6157-6162,Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381, and Marks et al.,1991, J. Mol. Biol., 222:581). Techniques for the generation and use ofantibody phage libraries are also described in U.S. Pat. Nos. 5,969,108,6,172,197, 5,885,793, 6,521,404; 6,544,731; 6,555,313; 6,582,915;6,593,081; 6,300,064; 6,653,068; 6,706,484; and 7,264,963; and Rothe etal., 2007, J. Mol. Bio., 376:1182 (each of which is incorporated byreference in its entirety). Affinity maturation strategies and chainshuffling strategies (Marks et al., 1992, Bio/Technology 10:779-783,incorporated by reference in its entirety) are known in the art and canbe employed to generate high affinity human antibodies.

Humanized antibodies can also be made in transgenic mice containinghuman immunoglobulin loci that are capable upon immunization ofproducing the full repertoire of human antibodies in the absence ofendogenous immunoglobulin production. This approach is described in U.S.Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and5,661,016.

This invention also encompasses bispecific antibodies that specificallyrecognize a CD37. Bispecific antibodies are antibodies that are capableof specifically recognizing and binding at least two different epitopes.The different epitopes can either be within the same molecule (e.g. thesame CD37) or on different molecules such that both, for example, theantibodies can specifically recognize and bind a CD37 as well as, forexample, 1) an effector molecule on a leukocyte such as a T-cellreceptor (e.g. CD3) or Fc receptor (e.g. CD64, CD32, or CD16) or 2) acytotoxic agent as described in detail below.

Exemplary bispecific antibodies can bind to two different epitopes, atleast one of which originates in a polypeptide of the invention.Alternatively, an anti-antigenic arm of an immunoglobulin molecule canbe combined with an arm which binds to a triggering molecule on aleukocyte such as a T cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG so as to focus cellular defense mechanismsto the cell expressing the particular antigen. Bispecific antibodies canalso be used to direct cytotoxic agents to cells which express aparticular antigen. These antibodies possess an antigen-binding arm andan arm which binds a cytotoxic agent or a radionuclide chelator, such asEOTUBE, DPTA, DOTA, or TETA. Techniques for making bispecific antibodiesare common in the art (Millstein et al., 1983, Nature 305:537-539;Brennan et al., 1985, Science 229:81; Suresh et al, 1986, Methods inEnzymol. 121:120; Traunecker et al., 1991, EMBO J. 10:3655-3659; Shalabyet al., 1992, J. Exp. Med. 175:217-225; Kostelny et al., 1992, J.Immunol. 148:1547-1553; Gruber et al., 1994, J. Immunol. 152:5368; andU.S. Pat. No. 5,731,168). Antibodies with more than two valencies arealso contemplated. For example, trispecific antibodies can be prepared(Tutt et al., J. Immunol. 147:60 (1991)). Thus, in certain embodimentsthe antibodies to CD37 are multispecific.

In certain embodiments are provided an antibody fragment to, forexample, increase tissue penetration. Various techniques are known forthe production of antibody fragments. Traditionally, these fragments arederived via proteolytic digestion of intact antibodies (for exampleMorimoto et al., 1993, Journal of Biochemical and Biophysical Methods24:107-117; Brennan et al., 1985, Science, 229:81). In certainembodiments, antibody fragments are produced recombinantly. Fab, Fv, andscFv antibody fragments can all be expressed in and secreted from E.coli or other host cells, thus allowing the production of large amountsof these fragments. Such antibody fragments can also be isolated fromthe antibody phage libraries discussed above. The antibody fragment canalso be linear antibodies as described in U.S. Pat. No. 5,641,870, forexample, and can be monospecific or bispecific. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner.

According to the present invention, techniques can be adapted for theproduction of single-chain antibodies specific to CD37 (see U.S. Pat.No. 4,946,778). In addition, methods can be adapted for the constructionof Fab expression libraries (Huse, et al., Science 246:1275-1281 (1989))to allow rapid and effective identification of monoclonal Fab fragmentswith the desired specificity for CD37, or derivatives, fragments,analogs or homologs thereof. Antibody fragments can be produced bytechniques in the art including, but not limited to: (a) a F(ab′)2fragment produced by pepsin digestion of an antibody molecule; (b) a Fabfragment generated by reducing the disulfide bridges of an F(ab′)2fragment, (c) a Fab fragment generated by the treatment of the antibodymolecule with papain and a reducing agent, and (d) Fv fragments.

It can further be desirable, especially in the case of antibodyfragments, to modify an antibody in order to increase its serumhalf-life. This can be achieved, for example, by incorporation of asalvage receptor binding epitope into the antibody fragment by mutationof the appropriate region in the antibody fragment or by incorporatingthe epitope into a peptide tag that is then fused to the antibodyfragment at either end or in the middle (e.g., by DNA or peptidesynthesis).

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune cells to unwanted cells (U.S. Pat. No. 4,676,980). It iscontemplated that the antibodies can be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins can be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

For the purposes of the present invention, it should be appreciated thatmodified antibodies can comprise any type of variable region thatprovides for the association of the antibody with the polypeptides of ahuman CD37. In this regard, the variable region can comprise or bederived from any type of mammal that can be induced to mount a humoralresponse and generate immunoglobulins against the desired antigen. Assuch, the variable region of the modified antibodies can be, forexample, of human, murine, non-human primate (e.g. cynomolgus monkeys,macaques, etc.) or lupine origin. In some embodiments both the variableand constant regions of the modified immunoglobulins are human. In otherembodiments the variable regions of compatible antibodies (usuallyderived from a non-human source) can be engineered or specificallytailored to improve the binding properties or reduce the immunogenicityof the molecule. In this respect, variable regions useful in the presentinvention can be humanized or otherwise altered through the inclusion ofimported amino acid sequences.

In certain embodiments, the variable domains in both the heavy and lightchains are altered by at least partial replacement of one or more CDRsand, if necessary, by partial framework region replacement and sequencechanging. Although the CDRs can be derived from an antibody of the sameclass or even subclass as the antibody from which the framework regionsare derived, it is envisaged that the CDRs will be derived from anantibody of different class and possibly from an antibody from adifferent species. It is not always necessary to replace all of the CDRswith the complete CDRs from the donor variable region to transfer theantigen binding capacity of one variable domain to another. Rather, insome cases it is only necessary to transfer those residues that arenecessary to maintain the activity of the antigen binding site. Giventhe explanations set forth in U.S. Pat. Nos. 5,585,089, 5,693,761 and5,693,762, it will be well within the competence of those skilled in theart, either by carrying out routine experimentation or by trial anderror testing to obtain a functional antibody with reducedimmunogenicity.

Alterations to the variable region notwithstanding, those skilled in theart will appreciate that the modified antibodies of this invention willcomprise antibodies (e.g., full-length antibodies or immunoreactivefragments thereof) in which at least a fraction of one or more of theconstant region domains has been deleted or otherwise altered so as toprovide desired biochemical characteristics such as reduced serumhalf-life when compared with an antibody of approximately the sameimmunogenicity comprising a native or unaltered constant region. In someembodiments, the constant region of the modified antibodies willcomprise a human constant region. Modifications to the constant regioncompatible with this invention comprise additions, deletions orsubstitutions of one or more amino acids in one or more domains. Thatis, the modified antibodies disclosed herein can comprise alterations ormodifications to one or more of the three heavy chain constant domains(CH1, CH2 or CH3) and/or to the light chain constant domain (CL). Insome embodiments, modified constant regions wherein one or more domainsare partially or entirely deleted are contemplated. In some embodiments,the modified antibodies will comprise domain deleted constructs orvariants wherein the entire CH2 domain has been removed (ΔCH2constructs). In some embodiments, the omitted constant region domainwill be replaced by a short amino acid spacer (e.g. 10 residues) thatprovides some of the molecular flexibility typically imparted by theabsent constant region.

Besides their configuration, it is known in the art that the constantregion mediates several effector functions. For example, binding of theC1 component of complement to antibodies activates the complementsystem. Activation of complement is important in the opsonisation andlysis of cell pathogens. The activation of complement also stimulatesthe inflammatory response and can also be involved in autoimmunehypersensitivity. Further, antibodies bind to cells via the Fc region,with a Fc receptor site on the antibody Fc region binding to a Fcreceptor (FcR) on a cell. There are a number of Fc receptors which arespecific for different classes of antibody, including IgG (gammareceptors), IgE (eta receptors), IgA (alpha receptors) and IgM (mureceptors). Binding of antibody to Fc receptors on cell surfacestriggers a number of important and diverse biological responsesincluding engulfment and destruction of antibody-coated particles,clearance of immune complexes, lysis of antibody-coated target cells bykiller cells (called antibody-dependent cell-mediated cytotoxicity, orADCC), release of inflammatory mediators, placental transfer and controlof immunoglobulin production.

In certain embodiments, the CD37-binding antibodies provide for alteredeffector functions that, in turn, affect the biological profile of theadministered antibody. For example, the deletion or inactivation(through point mutations or other means) of a constant region domain canreduce Fc receptor binding of the circulating modified antibody. Inother cases, it can be that constant region modifications, consistentwith this invention, moderate complement binding and thus reduce theserum half-life and nonspecific association of a conjugated cytotoxin.Yet other modifications of the constant region can be used to eliminatedisulfide linkages or oligosaccharide moieties that allow for enhancedlocalization due to increased antigen specificity or antibodyflexibility. Similarly, modifications to the constant region inaccordance with this invention can easily be made using well knownbiochemical or molecular engineering techniques well within the purviewof the skilled artisan.

In certain embodiments, a CD37-binding agent that is an antibody doesnot have one or more effector functions. For instance, in someembodiments, the antibody has no antibody-dependent cellularcytotoxicity (ADCC) activity and/or no complement-dependent cytotoxicity(CDC) activity. In certain embodiments, the antibody does not bind to anFc receptor and/or complement factors. In certain embodiments, theantibody has no effector function.

It will be noted that in certain embodiments, the modified antibodiescan be engineered to fuse the CH3 domain directly to the hinge region ofthe respective modified antibodies. In other constructs it can bedesirable to provide a peptide spacer between the hinge region and themodified CH2 and/or CH3 domains. For example, compatible constructscould be expressed wherein the CH2 domain has been deleted and theremaining CH3 domain (modified or unmodified) is joined to the hingeregion with a 5-20 amino acid spacer. Such a spacer can be added, forinstance, to ensure that the regulatory elements of the constant domainremain free and accessible or that the hinge region remains flexible.However, it should be noted that amino acid spacers can, in some cases,prove to be immunogenic and elicit an unwanted immune response againstthe construct. Accordingly, in certain embodiments, any spacer added tothe construct will be relatively non-immunogenic, or even omittedaltogether, so as to maintain the desired biochemical qualities of themodified antibodies.

Besides the deletion of whole constant region domains, it will beappreciated that the antibodies of the present invention can be providedby the partial deletion or substitution of a few or even a single aminoacid. For example, the mutation of a single amino acid in selected areasof the CH2 domain can be enough to substantially reduce Fc binding.Similarly, it can be desirable to simply delete that part of one or moreconstant region domains that control the effector function (e.g.complement CLQ binding) to be modulated. Such partial deletions of theconstant regions can improve selected characteristics of the antibody(serum half-life) while leaving other desirable functions associatedwith the subject constant region domain intact. Moreover, as alluded toabove, the constant regions of the disclosed antibodies can be modifiedthrough the mutation or substitution of one or more amino acids thatenhances the profile of the resulting construct. In this respect it canbe possible to disrupt the activity provided by a conserved binding site(e.g. Fc binding) while substantially maintaining the configuration andimmunogenic profile of the modified antibody. Certain embodiments cancomprise the addition of one or more amino acids to the constant regionto enhance desirable characteristics such as decreasing or increasingeffector function or provide for more cytotoxin or carbohydrateattachment. In such embodiments it can be desirable to insert orreplicate specific sequences derived from selected constant regiondomains.

The present invention further embraces variants and equivalents whichare substantially homologous to the chimeric, humanized and humanantibodies, or antibody fragments thereof, set forth herein. These cancontain, for example, conservative substitution mutations, i.e. thesubstitution of one or more amino acids by similar amino acids. Forexample, conservative substitution refers to the substitution of anamino acid with another within the same general class such as, forexample, one acidic amino acid with another acidic amino acid, one basicamino acid with another basic amino acid or one neutral amino acid byanother neutral amino acid. What is intended by a conservative aminoacid substitution is well known in the art.

The polypeptides of the present invention can be recombinantpolypeptides, natural polypeptides, or synthetic polypeptides comprisingan antibody, or fragment thereof, against a human CD37. It will berecognized in the art that some amino acid sequences of the inventioncan be varied without significant effect of the structure or function ofthe protein. Thus, the invention further includes variations of thepolypeptides which show substantial activity or which include regions ofan antibody, or fragment thereof, against CD37 protein. Such mutantsinclude deletions, insertions, inversions, repeats, and typesubstitutions.

The polypeptides and analogs can be further modified to containadditional chemical moieties not normally part of the protein. Thosederivatized moieties can improve the solubility, the biologicalhalf-life or absorption of the protein. The moieties can also reduce oreliminate any desirable side effects of the proteins and the like. Anoverview for those moieties can be found in REMINGTON'S PHARMACEUTICALSCIENCES, 20th ed., Mack Publishing Co., Easton, Pa. (2000).

The isolated polypeptides described herein can be produced by anysuitable method known in the art. Such methods range from direct proteinsynthetic methods to constructing a DNA sequence encoding isolatedpolypeptide sequences and expressing those sequences in a suitabletransformed host. In some embodiments, a DNA sequence is constructedusing recombinant technology by isolating or synthesizing a DNA sequenceencoding a wild-type protein of interest. Optionally, the sequence canbe mutagenized by site-specific mutagenesis to provide functionalanalogs thereof. See, e.g. Zoeller et al., Proc. Nat'l. Acad. Sci. USA81:5662-5066 (1984) and U.S. Pat. No. 4,588,585.

In some embodiments a DNA sequence encoding a polypeptide of interestwould be constructed by chemical synthesis using an oligonucleotidesynthesizer. Such oligonucleotides can be designed based on the aminoacid sequence of the desired polypeptide and selecting those codons thatare favored in the host cell in which the recombinant polypeptide ofinterest will be produced. Standard methods can be applied to synthesizean isolated polynucleotide sequence encoding an isolated polypeptide ofinterest. For example, a complete amino acid sequence can be used toconstruct a back-translated gene. Further, a DNA oligomer containing anucleotide sequence coding for the particular isolated polypeptide canbe synthesized. For example, several small oligonucleotides coding forportions of the desired polypeptide can be synthesized and then ligated.The individual oligonucleotides typically contain 5′ or 3′ overhangs forcomplementary assembly.

Once assembled (by synthesis, site-directed mutagenesis or anothermethod), the polynucleotide sequences encoding a particular isolatedpolypeptide of interest will be inserted into an expression vector andoperatively linked to an expression control sequence appropriate forexpression of the protein in a desired host. Proper assembly can beconfirmed by nucleotide sequencing, restriction mapping, and expressionof a biologically active polypeptide in a suitable host. As is wellknown in the art, in order to obtain high expression levels of atransfected gene in a host, the gene must be operatively linked totranscriptional and translational expression control sequences that arefunctional in the chosen expression host.

In certain embodiments, recombinant expression vectors are used toamplify and express DNA encoding antibodies, or fragments thereof,against human CD37. Recombinant expression vectors are replicable DNAconstructs which have synthetic or cDNA-derived DNA fragments encoding apolypeptide chain of an anti-CD37 antibody, or fragment thereof,operatively linked to suitable transcriptional or translationalregulatory elements derived from mammalian, microbial, viral or insectgenes. A transcriptional unit generally comprises an assembly of (1) agenetic element or elements having a regulatory role in gene expression,for example, transcriptional promoters or enhancers, (2) a structural orcoding sequence which is transcribed into mRNA and translated intoprotein, and (3) appropriate transcription and translation initiationand termination sequences, as described in detail below. Such regulatoryelements can include an operator sequence to control transcription. Theability to replicate in a host, usually conferred by an origin ofreplication, and a selection gene to facilitate recognition oftransformants can additionally be incorporated. DNA regions areoperatively linked when they are functionally related to each other. Forexample, DNA for a signal peptide (secretory leader) is operativelylinked to DNA for a polypeptide if it is expressed as a precursor whichparticipates in the secretion of the polypeptide; a promoter isoperatively linked to a coding sequence if it controls the transcriptionof the sequence; or a ribosome binding site is operatively linked to acoding sequence if it is positioned so as to permit translation.Structural elements intended for use in yeast expression systems includea leader sequence enabling extracellular secretion of translated proteinby a host cell. Alternatively, where recombinant protein is expressedwithout a leader or transport sequence, it can include an N-terminalmethionine residue. This residue can optionally be subsequently cleavedfrom the expressed recombinant protein to provide a final product.

The choice of expression control sequence and expression vector willdepend upon the choice of host. A wide variety of expression host/vectorcombinations can be employed. Useful expression vectors for eukaryotichosts, include, for example, vectors comprising expression controlsequences from SV40, bovine papilloma virus, adenovirus andcytomegalovirus. Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from Esherichia coli,including pCR 1, pBR322, pMB9 and their derivatives, wider host rangeplasmids, such as M13 and filamentous single-stranded DNA phages.

Suitable host cells for expression of a CD37-binding polypeptide orantibody (or a CD37 protein to use as an antigen) include prokaryotes,yeast, insect or higher eukaryotic cells under the control ofappropriate promoters. Prokaryotes include gram negative or grampositive organisms, for example E. coli or bacilli. Higher eukaryoticcells include established cell lines of mammalian origin as describedbelow. Cell-free translation systems could also be employed. Appropriatecloning and expression vectors for use with bacterial, fungal, yeast,and mammalian cellular hosts are described by Pouwels et al. (CloningVectors: A Laboratory Manual, Elsevier, N.Y., 1985), the relevantdisclosure of which is hereby incorporated by reference. Additionalinformation regarding methods of protein production, including antibodyproduction, can be found, e.g., in U.S. Patent Publication No.2008/0187954, U.S. Pat. Nos. 6,413,746 and 6,660,501, and InternationalPatent Publication No. WO 04009823, each of which is hereby incorporatedby reference herein in its entirety.

Various mammalian or insect cell culture systems are also advantageouslyemployed to express recombinant protein. Expression of recombinantproteins in mammalian cells can be performed because such proteins aregenerally correctly folded, appropriately modified and completelyfunctional. Examples of suitable mammalian host cell lines include theCOS-7 lines of monkey kidney cells, described by Gluzman (Cell 23:175,1981), and other cell lines capable of expressing an appropriate vectorincluding, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO),HeLa and BHK cell lines. Mammalian expression vectors can comprisenontranscribed elements such as an origin of replication, a suitablepromoter and enhancer linked to the gene to be expressed, and other 5′or 3′ flanking nontranscribed sequences, and 5′ or 3′ nontranslatedsequences, such as necessary ribosome binding sites, a polyadenylationsite, splice donor and acceptor sites, and transcriptional terminationsequences. Baculovirus systems for production of heterologous proteinsin insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47(1988).

The proteins produced by a transformed host can be purified according toany suitable method. Such standard methods include chromatography (e.g.,ion exchange, affinity and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for protein purification. Affinity tags such as hexahistidine,maltose binding domain, influenza coat sequence andglutathione-S-transferase can be attached to the protein to allow easypurification by passage over an appropriate affinity column. Isolatedproteins can also be physically characterized using such techniques asproteolysis, nuclear magnetic resonance and x-ray crystallography.

For example, supernatants from systems which secrete recombinant proteininto culture media can be first concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore Pellicon ultrafiltration unit. Following the concentrationstep, the concentrate can be applied to a suitable purification matrix.Alternatively, an anion exchange resin can be employed, for example, amatrix or substrate having pendant diethylaminoethyl (DEAE) groups. Thematrices can be acrylamide, agarose, dextran, cellulose or other typescommonly employed in protein purification. Alternatively, a cationexchange step can be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a CD37-binding agent. Some or all of theforegoing purification steps, in various combinations, can also beemployed to provide a homogeneous recombinant protein.

Recombinant protein produced in bacterial culture can be isolated, forexample, by initial extraction from cell pellets, followed by one ormore concentration, salting-out, aqueous ion exchange or size exclusionchromatography steps. High performance liquid chromatography (HPLC) canbe employed for final purification steps. Microbial cells employed inexpression of a recombinant protein can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

Methods known in the art for purifying antibodies and other proteinsalso include, for example, those described in U.S. Patent PublicationNo. 2008/0312425, 2008/0177048, and 2009/0187005, each of which ishereby incorporated by reference herein in its entirety.

In certain embodiments, the CD37-binding agent is a polypeptide that isnot an antibody. A variety of methods for identifying and producingnon-antibody polypeptides that bind with high affinity to a proteintarget are known in the art. See e.g., Skerra, Curr. Opin. Biotechnol.,18:295-304 (2007), Hosse et al., Protein Science, 15:14-27 (2006), Gillet al., Curr. Opin. Biotechnol., 17:653-658 (2006), Nygren, FEBS J.,275:2668-76 (2008), and Skerra, FEBS J., 275:2677-83 (2008), each ofwhich is incorporated by reference herein in its entirety. In certainembodiments, phage display technology has been used to identify/producethe CD37-binding polypeptide. In certain embodiments, the polypeptidecomprises a protein scaffold of a type selected from the groupconsisting of protein A, a lipocalin, a fibronectin domain, an ankyrinconsensus repeat domain, and thioredoxin.

In some embodiments, the agent is a non-protein molecule. In certainembodiments, the agent is a small molecule. Combinatorial chemistrylibraries and techniques useful in the identification of non-proteinCD37-binding agents are known to those skilled in the art. See, e.g.,Kennedy et al., J. Comb. Chem, 10:345-354 (2008), Dolle et al, J. Comb.Chem., 9:855-902 (2007), and Bhattacharyya, Curr. Med. Chem., 8:1383-404(2001), each of which is incorporated by reference herein in itsentirety. In certain further embodiments, the agent is a carbohydrate, aglycosaminoglycan, a glycoprotein, or a proteoglycan.

In certain embodiments, the agent is a nucleic acid aptamer. Aptamersare polynucleotide molecules that have been selected (e.g., from randomor mutagenized pools) on the basis of their ability to bind to anothermolecule. In some embodiments, the aptamer comprises a DNApolynucleotide. In certain alternative embodiments, the aptamercomprises an RNA polynucleotide. In certain embodiments, the aptamercomprises one or more modified nucleic acid residues. Methods ofgenerating and screening nucleic acid aptamers for binding to proteinsare well known in the art. See, e.g., U.S. Pat. Nos. 5,270,163,5,683,867, 5,763,595, 6,344,321, 7,368,236, 5,582,981, 5,756,291,5,840,867, 7,312,325, 7,329,742, International Patent Publication No. WO02/077262, International Patent Publication No. WO 03/070984, U.S.Patent Application Publication No. 2005/0239134, U.S. Patent ApplicationPublication No. 2005/0124565, and U.S. Patent Application PublicationNo. 2008/0227735, each of which is incorporated by reference herein inits entirety.

III. Immunoconjugates

The present invention is also directed to conjugates (also referred toherein as immunoconjugates), comprising the anti-CD37 antibodies,antibody fragments, and their functional equivalents as disclosedherein, linked or conjugated to a drug or prodrug. Suitable drugs orprodrugs are known in the art. The drugs or prodrugs can be cytotoxicagents. The cytotoxic agent used in the cytotoxic conjugate of thepresent invention can be any compound that results in the death of acell, or induces cell death, or in some manner decreases cell viability,and includes, for example, maytansinoids and maytansinoid analogs. Othersuitable cytotoxic agents are for example benzodiazepines, taxoids,CC-1065 and CC-1065 analogs, duocarmycins and duocarmycin analogs,enediynes, such as calicheamicins, dolastatin and dolastatin analogsincluding auristatins, tomaymycin derivatives, leptomycin derivatives,methotrexate, cisplatin, carboplatin, daunorubicin, doxorubicin,vincristine, vinblastine, melphalan, mitomycin C, chlorambucil andmorpholino doxorubicin.

Such conjugates can be prepared by using a linking group in order tolink a drug or prodrug to the antibody or functional equivalent.Suitable linking groups are well known in the art and include, forexample, disulfide groups, thioether groups, acid labile groups,photolabile groups, peptidase labile groups and esterase labile groups.

The drug or prodrug can, for example, be linked to the anti-CD37antibody or fragment thereof through a disulfide bond. The linkermolecule or crosslinking agent comprises a reactive chemical group thatcan react with the anti-CD37 antibody or fragment thereof. The reactivechemical groups for reaction with the cell-binding agent can beN-succinimidyl esters and N-sulfosuccinimidyl esters. Additionally thelinker molecule comprises a reactive chemical group, which can be adithiopyridyl group that can react with the drug to form a disulfidebond. Linker molecules include, for example, N-succinimidyl3-(2-pyridyldithio) propionate (SPDP) (see, e.g., Carlsson et al.,Biochem. J., 173: 723-737 (1978)), N-succinimidyl4-(2-pyridyldithio)butanoate (SPDB) (see, e.g., U.S. Pat. No.4,563,304), N-succinimidyl 4-(2-pyridyldithio)2-sulfobutanoate(sulfo-SPDB) (see US Publication No. 20090274713), N-succinimidyl4-(2-pyridyldithio) pentanoate (SPP) (see, e.g., CAS Registry number341498-08-6), 2-iminothiolane, or acetylsuccinic anhydride. For example,the antibody or cell binding agent can be modified with crosslinkingreagents and the antibody or cell binding agent containing free orprotected thiol groups thus derived is then reacted with a disulfide- orthiol-containing maytansinoid to produce conjugates. The conjugates canbe purified by chromatography, including but not limited to HPLC,size-exclusion, adsorption, ion exchange and affinity capture, dialysisor tangential flow filtration.

In another aspect of the present invention, the anti-CD37 antibody islinked to cytotoxic drugs via disulfide bonds and a polyethylene glycolspacer in enhancing the potency, solubility or the efficacy of theimmunoconjugate. Such cleavable hydrophilic linkers are described inWO2009/0134976. The additional benefit of this linker design is thedesired high monomer ratio and the minimal aggregation of theantibody-drug conjugate. Specifically contemplated in this aspect areconjugates of cell-binding agents and drugs linked via disulfide group(—S—S—) bearing polyethylene glycol spacers ((CH₂CH₂O)_(n=1-14)) with anarrow range of drug load of 2-8 are described that show relatively highpotent biological activity toward cells and have the desired biochemicalproperties of high conjugation yield and high monomer ratio with minimalprotein aggregation.

Specifically contemplated in this aspect is an anti-CD37 antibody drugconjugate of formula (I) or a conjugate of formula (I′):CB—[X₁—(—CH₂—CH₂O—)_(n)—Y-D]_(m)  (I)[D-Y—(—CH₂—CH₂O—)_(n)—X_(l)]_(m)—CB  (I′)wherein:

CB represents an anti-CD37 antibody or fragment;

D represents a drug;

X represents an aliphatic, an aromatic or a heterocyclic unit attachedto the cell-binding agent via a thioether bond, an amide bond, acarbamate bond, or an ether bond;

Y represents an aliphatic, an aromatic or a heterocyclic unit attachedto the drug via a disulfide bond;

l is 0 or 1;

m is an integer from 2 to 8; and

n is an integer from 1 to 24.

In some embodiments, m is an integer from 2 to 6.

In some embodiments, m is an integer from 3 to 5.

In some embodiments, n is an integer form 2 to 8. Alternatively, asdisclosed in, for example, U.S. Pat. Nos. 6,441,163 and 7,368,565, thedrug can be first modified to introduce a reactive ester suitable toreact with a cell-binding agent. Reaction of these drugs containing anactivated linker moiety with a cell-binding agent provides anothermethod of producing a cell-binding agent drug conjugate. Maytansinoidscan also be linked to anti-CD37 antibody or fragment using PEG linkinggroups, as set forth for example in U.S. Pat. No. 6,716,821. These PEGnon-cleavable linking groups are soluble both in water and innon-aqueous solvents, and can be used to join one or more cytotoxicagents to a cell binding agent. Exemplary PEG linking groups includeheterobifunctional PEG linkers that react with cytotoxic agents and cellbinding agents at opposite ends of the linkers through a functionalsulfhydryl or disulfide group at one end, and an active ester at theother end. As a general example of the synthesis of a cytotoxicconjugate using a PEG linking group, reference is again made to U.S.Pat. No. 6,716,821 which is incorporated entirely by reference herein.Synthesis begins with the reaction of one or more cytotoxic agentsbearing a reactive PEG moiety with a cell-binding agent, resulting indisplacement of the terminal active ester of each reactive PEG moiety byan amino acid residue of the cell binding agent, to yield a cytotoxicconjugate comprising one or more cytotoxic agents covalently bonded to acell binding agent through a PEG linking group. Alternatively, the cellbinding can be modified with the bifunctional PEG crosslinker tointroduce a reactive disulfide moiety (such as a pyridyldisulfide),which can then be treated with a thiol-containing maytansinoid toprovide a conjugate. In another method, the cell binding can be modifiedwith the bifunctional PEG crosslinker to introduce a thiol moiety whichcan then can be treated with a reactive disulfide-containingmaytansinoid (such as a pyridyldisulfide), to provide a conjugate.

Antibody-maytansinoid conjugates with non-cleavable links can also beprepared. Such crosslinkers are described in the art (see US PublicationNo. 20050169933) and include but are not limited to, N-succinimidyl4-(maleimidomethyl) cyclohexanecarboxylate (SMCC). In some embodiments,the antibody is modified with crosslinking reagents such as succinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), sulfo-SMCC,maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), sulfo-MBS orsuccinimidyl-iodoacetate, as described in the literature, to introduce1-10 reactive groups (Yoshitake et al, Eur. J. Biochem., 101:395-399(1979); Hashida et al, J. Applied Biochem., 56-63 (1984); and Liu et al,Biochem., 18:690-697 (1979)). The modified antibody is then reacted withthe thiol-containing maytansinoid derivative to produce a conjugate. Theconjugate can be purified by gel filtration through a Sephadex G25column or by dialysis or tangential flow filtration. The modifiedantibodies are treated with the thiol-containing maytansinoid (1 to 2molar equivalent/maleimido group) and antibody-maytansinoid conjugatesare purified by gel filtration through a Sephadex G-25 column,chromatography on a ceramic hydroxyapatite column, dialysis ortangential flow filtration or a combination of methods thereof.Typically, an average of 1-10 maytansinoids per antibody are linked. Onemethod is to modify antibodies with succinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) to introducemaleimido groups followed by reaction of the modified antibody with athiol-containing maytansinoid to give a thioether-linked conjugate.Again conjugates with 1 to 10 drug molecules per antibody moleculeresult. Maytansinoid conjugates of antibodies, antibody fragments, andother proteins are made in the same way.

In another aspect of the invention, the CD37 antibody is linked to thedrug via a non-cleavable bond through the intermediacy of a PEG spacer.Suitable crosslinking reagents comprising hydrophilic PEG chains thatform linkers between a drug and the anti-CD37 antibody or fragment arealso well known in the art, or are commercially available (for examplefrom Quanta Biodesign, Powell, Ohio). Suitable PEG-containingcrosslinkers can also be synthesized from commercially available PEGsthemselves using standard synthetic chemistry techniques known to oneskilled in the art. The drugs can be reacted with bifunctionalPEG-containing cross linkers to give compounds of the following formula,Z—X₁—(—CH₂—CH₂—O—)_(n)—Y_(p)-D, by methods described in detail in USPatent Publication 20090274713 and in WO2009/0134976, which can thenreact with the cell binding agent to provide a conjugate. Alternatively,the cell binding can be modified with the bifunctional PEG crosslinkerto introduce a thiol-reactive group (such as a maleimide orhaloacetamide) which can then be treated with a thiol-containingmaytansinoid to provide a conjugate. In another method, the cell bindingcan be modified with the bifunctional PEG crosslinker to introduce athiol moiety which can then be treated with a thiol-reactivemaytansinoid (such as a maytansinoid bearing a maleimide orhaloacetamide), to provide a conjugate.

Accordingly, another aspect of the present invention is an anti-CD37antibody drug conjugate of formula (II) or of formula (II′):CB—[X₁—(—CH₂—CH₂—O—)_(n)—Y_(p)-D]_(m)  (II)[D-Y_(p)—(—CH₂—CH₂—O—)_(n)—X_(l)]_(m)—CB  (II′)wherein, CB represents an anti-CD37 antibody or fragment;

D represents a drug;

X represents an aliphatic, an aromatic or a heterocyclic unit bonded tothe cell-binding agent via a thioether bond, an amide bond, a carbamatebond, or an ether bond;

Y represents an aliphatic, an aromatic, or a heterocyclic unit bonded tothe drug via a covalent bond selected from the group consisting of athioether bond, an amide bond, a carbamate bond, an ether bond, an aminebond, a carbon-carbon bond and a hydrazone bond;

l is 0 or 1;

p is 0 or 1;

m is an integer from 2 to 15; and

n is an integer from 1 to 2000.

In some embodiments, m is an integer from 2 to 8; and

In some embodiments, n is an integer from 1 to 24.

In some embodiments, m is an integer from 2 to 6.

In some embodiments, m is an integer from 3 to 5.

In some embodiments, n is an integer from 2 to 8. Examples of suitablePEG-containing linkers include linkers having an N-succinimidyl ester orN-sulfosuccinimidyl ester moiety for reaction with the anti-CD37antibody or fragment thereof, as well as a maleimido- orhaloacetyl-based moiety for reaction with the compound. A PEG spacer canbe incorporated into any crosslinker known in the art by the methodsdescribed herein.

Many of the linkers disclosed herein are described in detail in U.S.Patent Publication Nos. 20050169933 and 20090274713, and inWO2009/0134976; the contents of which are entirely incorporated hereinby reference.

The present invention includes aspects wherein about 2 to about 8 drugmolecules (“drug load”), for example, maytansinoid, are linked to ananti-CD37 antibody or fragment thereof “Drug load”, as used herein,refers to the number of drug molecules (e.g., a maytansinoid) that canbe attached to a cell binding agent (e.g., an anti-CD37 antibody orfragment thereof). In one aspect, the number of drug molecules that canbe attached to a cell binding agent can average from about 2 to about 8(e.g., 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5,4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9,6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3,7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1).N^(2′)-deacetyl-N^(2′)-(3-mercapto-1-oxopropyl)-maytansine (DM1) andN^(2′)-deacetyl-N^(2′)-(4-mercapto-4-methyl-1-oxopentyl) maytansine(DM4) can be used.

Thus, in one aspect, an immunoconjugate comprises 1 maytansinoid perantibody. In another aspect, an immunoconjugate comprises 2maytansinoids per antibody. In another aspect, an immunoconjugatecomprises 3 maytansinoids per antibody. In another aspect, animmunoconjugate comprises 4 maytansinoids per antibody. In anotheraspect, an immunoconjugate comprises 5 maytansinoids per antibody. Inanother aspect, an immunoconjugate comprises 6 maytansinoids perantibody. In another aspect, an immunoconjugate comprises 7maytansinoids per antibody. In another aspect, an immunoconjugatecomprises 8 maytansinoids per antibody.

In one aspect, an immunoconjugate comprises about 1 to about 8maytansinoids per antibody. In another aspect, an immunoconjugatecomprises about 2 to about 7 maytansinoids per antibody. In anotheraspect, an immunoconjugate comprises about 2 to about 6 maytansinoidsper antibody. In another aspect, an immunoconjugate comprises about 2 toabout 5 maytansinoids per antibody. In another aspect, animmunoconjugate comprises about 3 to about 5 maytansinoids per antibody.In another aspect, an immunoconjugate comprises about 3 to about 4maytansinoids per antibody.

In one aspect, a composition comprising immunoconjugates has an averageof about 2 to about 8 (e.g., 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0,4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4,5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8,6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1) drugmolecules (e.g., maytansinoids) attached per antibody. In one aspect, acomposition comprising immunoconjugates has an average of about 1 toabout 8 drug molecules (e.g., maytansinoids) per antibody. In oneaspect, a composition comprising immunoconjugates has an average ofabout 2 to about 7 drug molecules (e.g., maytansinoids) per antibody. Inone aspect, a composition comprising immunoconjugates has an average ofabout 2 to about 6 drug molecules (e.g., maytansinoids) per antibody. Inone aspect, a composition comprising immunoconjugates has an average ofabout 2 to about 5 drug molecules (e.g., maytansinoids) per antibody. Inone aspect, a composition comprising immunoconjugates has an average ofabout 3 to about 5 drug molecules (e.g., maytansinoids) per antibody. Inone aspect, a composition comprising immunoconjugates has an average ofabout 3 to about 4 drug molecules (e.g., maytansinoids) per antibody.

In one aspect, a composition comprising immunoconjugates has an averageof about 2±0.5, about 3±0.5, about 4±0.5, about 5±0.5, about 6±0.5,about 7±0.5, or about 8±0.5 drug molecules (e.g., maytansinoids)attached per antibody. In one aspect, a composition comprisingimmunoconjugates has an average of about 3.5±0.5 drug molecules (e.g.,maytansinoids) per antibody.

The anti-CD37 antibody or fragment thereof can be modified by reacting abifunctional crosslinking reagent with the anti-CD37 antibody orfragment thereof, thereby resulting in the covalent attachment of alinker molecule to the anti-CD37 antibody or fragment thereof. As usedherein, a “bifunctional crosslinking reagent” is any chemical moietythat covalently links a cell-binding agent to a drug, such as the drugsdescribed herein. In another method, a portion of the linking moiety isprovided by the drug. In this respect, the drug comprises a linkingmoiety that is part of a larger linker molecule that is used to join thecell-binding agent to the drug. For example, to form the maytansinoidDM1, the side chain at the C-3 hydroxyl group of maytansine is modifiedto have a free sulfhydryl group (SH). This thiolated form of maytansinecan react with a modified cell-binding agent to form a conjugate.Therefore, the final linker is assembled from two components, one ofwhich is provided by the crosslinking reagent, while the other isprovided by the side chain from DM1.

The drug molecules can also be linked to the antibody molecules throughan intermediary carrier molecule such as serum albumin.

As used herein, the expression “linked to a cell-binding agent” or“linked to an anti-CD37 antibody or fragment” refers to the conjugatemolecule comprising at least one drug derivative bound to a cell-bindingagent anti-CD37 antibody or fragment via a suitable linking group, or aprecursor thereof. One linking group is SMCC.

In certain embodiments, cytotoxic agents useful in the present inventionare maytansinoids and maytansinoid analogs. Examples of suitablemaytansinoids include esters of maytansinol and maytansinol analogs.Included are any drugs that inhibit microtubule formation and that arehighly toxic to mammalian cells, as are maytansinol and maytansinolanalogs.

Examples of suitable maytansinol esters include those having a modifiedaromatic ring and those having modifications at other positions. Suchsuitable maytansinoids are disclosed in U.S. Pat. Nos. 4,424,219;4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331,598;4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371,533;5,208,020; 5,416,064; 5,475,092; 5,585,499; 5,846,545; 6,333,410;7,276,497 and 7,473,796.

In a certain embodiment, the immunoconjugates of the invention utilizethe thiol-containing maytansinoid (DM1), formally termedN^(2′)-deacetyl-N^(2′)-(3-mercapto-1-oxopropyl)-maytansine, as thecytotoxic agent. DM1 is represented by the following structural formula(III):

In another embodiment, the conjugates of the present invention utilizethe thiol-containing maytansinoidN^(2′)-deacetyl-N^(2′)(4-methyl-4-mercapto-1-oxopentyl)-maytansine(e.g., DM4) as the cytotoxic agent. DM4 is represented by the followingstructural formula (IV):

Another maytansinoid comprising a side chain that contains a stericallyhindered thiol bond isN^(2′)-deacetyl-N-^(2′)(4-mercapto-1-oxopentyl)-maytansine (termed DM3),represented by the following structural formula (V):

Each of the maytansinoids taught in U.S. Pat. Nos. 5,208,020 and7,276,497, can also be used in the conjugate of the present invention.In this regard, the entire disclosure of U.S. Pat. Nos. 5,208,020 and7,276,697 is incorporated herein by reference.

Many positions on maytansinoids can serve as the position to chemicallylink the linking moiety. For example, the C-3 position having a hydroxylgroup, the C-14 position modified with hydroxymethyl, the C-15 positionmodified with hydroxy and the C-20 position having a hydroxy group areall expected to be useful. In some embodiments, the C-3 position servesas the position to chemically link the linking moiety, and in someparticular embodiments, the C-3 position of maytansinol serves as theposition to chemically link the linking moiety.

Structural representations of some conjugates are shown below:

Several descriptions for producing such antibody-maytansinoid conjugatesare provided in U.S. Pat. Nos. 6,333,410, 6,441,163, 6,716,821, and7,368,565, each of which is incorporated herein in its entirety.

In general, a solution of an antibody in aqueous buffer can be incubatedwith a molar excess of maytansinoids having a disulfide moiety thatbears a reactive group. The reaction mixture can be quenched by additionof excess amine (such as ethanolamine, taurine, etc.). Themaytansinoid-antibody conjugate can then be purified by gel filtration.

The number of maytansinoid molecules bound per antibody molecule can bedetermined by measuring spectrophotometrically the ratio of theabsorbance at 252 nm and 280 nm. The average number of maytansinoidmolecules/antibody can be, for example, about 1-10, 2-5, 3-4, or about3.5. In one aspect, the average number of maytansinoidmolecules/antibody is about 3.5±0.5.

Anthracycline compounds, as well as derivatives, intermediates andmodified versions thereof, can also be used to prepare anti-CD37immunoconjugates. For example, doxorubicin, doxorubicin derivatives,doxorubicin intermediates, and modified doxorubicins can be used inanti-CD37 conjugates. Exemplary compounds are described in WO2010/009124, which is herein incorporated by reference in its entirety.Such compounds include, for example, compounds of the following formula:

wherein R₁ is a hydrogen atom, hydroxy or methoxy group and R₂ is aC₁-C_(s) alkoxy group, or a pharmaceutically acceptable salt thereof.

Conjugates of antibodies with maytansinoid or other drugs can beevaluated for their ability to suppress proliferation of variousunwanted cell lines in vitro. For example, cell lines such as the humanlymphoma cell line Daudi and the human lymphoma cell line Ramos, caneasily be used for the assessment of cytotoxicity of these compounds.Cells to be evaluated can be exposed to the compounds for 4 to 5 daysand the surviving fractions of cells measured in direct assays by knownmethods. IC₅₀ values can then be calculated from the results of theassays.

The immunoconjugates can, according to some embodiments describedherein, be internalized into cells. The immunoconjugate, therefore, canexert a therapeutic effect when it is taken up by, or internalized, by aCD37-expressing cell. In some particular embodiments, theimmunoconjugate comprises an antibody, antibody fragment, orpolypeptide, linked to a cytotoxic agent by a cleavable linker, and thecytotoxic agent is cleaved from the antibody, antibody fragment, orpolypeptide, wherein it is internalized by a CD37-expressing cell.

In some embodiments, the immunoconjugates are capable of depletingB-cells, e.g. autoreactive B-cells. For example, in some embodiments,treatment with an immunoconjugate results in a depletion of at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, or at least about 75%of B-cells.

In another aspect of the invention siRNA molecules can be linked to theantibodies of the present invention instead of a drug. siRNAs can belinked to the antibodies of the present invention by methods commonlyused for the modification of oligonucleotides (see, for example, USPatent Publications 20050107325 and 20070213292). Thus the siRNA in its3′ or 5′-phosphoroamidite form can be reacted with one end of thecrosslinker bearing a hydroxyl functionality to give an ester bondbetween the siRNA and the crosslinker. Similarly reaction of the siRNAphosphoramidite with a crosslinker bearing a terminal amino groupresults in linkage of the crosslinker to the siRNA through an amine.Alternatively, the siRNA can be derivatized by standard chemical methodsto introduce a thiol group. This thiol-containing siRNA can be reactedwith an antibody, that has been modified to introduce an activedisulfide or maleimide moiety, to produce a cleavable or non-cleavableconjugate. Between 1-20 siRNA molecules can be linked to an antibody bythis method.

III. Polynucleotides

In certain embodiments, the invention encompasses polynucleotidescomprising polynucleotides that encode a polypeptide that specificallybinds CD37 or a fragment of such a polypeptide. For example, theinvention provides a polynucleotide comprising a nucleic acid sequencethat encodes an antibody to a human CD37 or encodes a fragment of suchan antibody. The polynucleotides of the invention can be in the form ofRNA or in the form of DNA. DNA includes cDNA, genomic DNA, and syntheticDNA; and can be double-stranded or single-stranded, and if singlestranded can be the coding strand or non-coding (anti-sense) strand.

In certain embodiments, the polynucleotides are isolated. In certainembodiments, the polynucleotides are substantially pure.

The invention provides a polynucleotide comprising a polynucleotideencoding a polypeptide comprising a sequence selected from the groupconsisting of SEQ ID NOs:4-120.

The invention further provides a polynucleotide comprising a sequenceselected from those shown in Tables 7-10 below.

TABLE 7  Variable heavy chain polynucleotide sequences AntibodyVH Polynucleotide Sequence (SEQ ID NO) muCD37-3caggtgcaggtgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccattacatgcactgtctcagggttctcattaaccacctctggtgtaagctgggttcgccagcctccaggaaagggtctggagtggctgggagtaatatggggtgacgggagcacaaactatcattcagctctcaaatccagactgagcatcaagaaggatcactccaagagccaagttttcttaaaactgaacagtctgcaaactgatgacacagccacgtactactgtgccaaaggaggctactcgttggctcactggggccaagggactctggtcacagtctctgca (SEQ ID NO: 121)chCD37-3aagcttgccaccatggctgtcctggcactgctcctctgcctggtgacatacccaagctgtgtcctatcacaggtgcaggtgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccattacatgcactgtctcagggttctcattaaccacctctggtgtaagctgggttcgccagcctccaggaaagggtctggagtggctgggagtaatatggggtgacgggagcacaaactatcattcagctctcaaatccagactgagcatcaagaaggatcactccaagagccaagttttcttaaaactgaacagtctgcaaactgatgacacagccacgtactactgtgccaaaggaggctactcgttggctcactggggccaagggactctggtcacagtctctgcagcctctacgaagggccc (SEQ ID NO: 122) huCD37-3v1.0aagcttgccaccatgggttggagctgcattattctgtttctggtggccaccgccaccggtgtgcactcacaagtccaagtccaagaatctggtccaggtctggtggccccttcccaaactctgagcatcacctgtaccgtttctggttttagccttaccacctctggtgtgagttgggtacgccaaccacccggtaagggtctcgaatggctgggtgtaatctggggtgatggttccacaaattaccatccttccctcaagtcccgccttagcatcaaaaaggatcacagcaaaagtcaagttttcctgaaactgaatagtctgacagcagccgatacagccacctactattgcgccaagggtggttatagtcttgcacactggggtcaaggtaccctcgttaccgtctcctcagctagtaccaagggccc (SEQ ID NO: 123) huCD37-3v1.1aagcttgccaccatgggctggagctgtatcattctgtactggtggcgacagctactggggtccactcccaagtgcaggtacaagagtccgggcctggattggtcgcaccaagccagaccctctctatcacttgtaccgttagcgggttctctctgacaaccagtggagtgagttgggtgaggcagccaccaggaaagggactggagtggctgggggtgataggggcgacggcagcacaaactatcattccagtcttaaatctcggttgtccattaaaaaagaccatagtaaatctcaagttttcctgaaactcaatagcctgacagccgcagacactgctacgtattactgcgccaaaggaggatacagtctggctcactggggacaggggaccctggtgaccgtgtcatccgcatcaacaaagggccc (SEQ ID NO: 124) muCD37-12cagatccagttggtgcagtctggacctgagctgaagaagcctggagagacagtcaagatctcctgcaaggcttctgggtataccttcacaaagtatggaatgaactgggtgaagcaggctcaaggaaagggtttaaagtggatgggctggataaacaccaacactggagagtcaagaaatgctgaagaattcaagggacggtagccttctctaggaaacctctgccagcactgcctatttgcagatcaacaacctcaaatatgaggacacggctacatatttctgtggaaggggcacggtagtagcggactggggccaaggcaccactctcacagtctcctca (SEQ ID NO: 125)chCD37-12aagcttgccaccatggggtggtcatgcataatcctctactggtcgctactgctaccggtgtgcactcacagattcagctggttcaaagtggcccagagctgaaaaagccaggggaaacagtgaaaataagttgcaaggcatccggttacactacacaaagtacggcatgaactgggtcaagcaggcccagggcaaggggctcaaatggatgggttggatcaataccaacactggcgagtctaggaatgctgaggagtttaagggccggtttgccttcagcctggagacaagtgccagcacagcttacctgcaaatcaacaatctgaagtatgaggatacagcaacctatactgcggccgcggcactgtcgttgcagactggggacaaggtaccaccttgactgtatccagtgccagcactaagggccc (SEQ ID NO: 126) muCD37-38gatgtgcagcttcaggagtcaggacctgacctggtgaaaccttctcagtcactacactcacctgcactgtcactggctactccatcaccagtggttttggctggcactggatccggcagtttccaggaaacaagctggaatggatggcctacatactctacagtggtggcactgactacaacccatctctcaaaagtcgaatctctatcactcgagacacttccaagaaccagttcttcctgcggttgagttctgtgactactgaggacacagccacatattactgtgcaagaggctactatggttacggggcctggtttgtttactggggccaagggactctggtcactgtctctgca (SEQ ID NO: 127) chCD37-38aagcttgccaccatgggctggagttgtatcattctgtttttggtggccaccgccactggagtccattcccaagtgcaactccaggaatctggccctgacctggttaagccatctcagagcctctccctgacctgcactgttacaggatactcaatcacatcaggctttggctggcactggatcagacaatttcccgggaacaagttggaatggatggcttacattctgtatagcgggggtaccgattacaatccttccctcaagagccgaatctctatcaccagggatacaagcaagaaccaattttttctccgcctcagctctgtgactaccgaagataccgctacttactattgtgccaggggctactatggatatggtgcatggttcgtctattggggccagggaaccctggtgactgtgagcgctgcctctaccaagggccc (SEQ ID NO: 128) huCD37-38aagcttgccaccatgggttggagctgcatcattcttttcctggtcgctactgcaactggagtccactcacaggtccagctgcaagagtccggtcctgggcttgtgaaacccagccagtccctcagtctcacctgtactgtctctggctactctattaccagtgggttcggctggcattggattaggcagtttcccggtaaggggctggagtggatggcatatatcctgtacagcggaggaaccgattacaacccaagtctgaagagcaggatcagcattacccgggacacaagcaaaaaccagtttttccttcggctgtctagtgttacagctgcagacaccgctacttactattgtgctcggggttactatggctatggggcttggtttgtgtattggggacaaggcactcttgtgaccgtgagcagcgcctcaacaaagggccc (SEQ ID NO: 129) muCD37-50gatgtgcagcttcaggagtcaggacctgacctgttgaaaccttctcagtcactacactcacctgcactgtcactggctactccatcaccagtggttagcctggcactggatccggcagtaccaggaaacaaactggaatggatgggctacatactctacagtggtagcactgtctacagcccatctctcaaaagtcgaatctctatcactcgagacacatccaagaaccacttcttcctgcagttgaattctgtgactactgaggacacagccacatattactgtgcaagagggtactatggttacggcgcctggtagcttactggggccaagggactctggtcactgtctctgca (SEQ ID NO: 130) huCD37-50aagcttgccaccatggggtggtcctgcataatccttttcctggttgctactgctaccggagtccattcacaggtgcagctgcaggagtccggccccggcctgctcaagccttctcagagtctgagtctgacttgtactgtactggctacagcataaccagcggtttcgcttggcactggatcagacagcatcccggcaacaaactggagtggatgggatacatactgtactcaggctcaactgtctattccccctccctgaaatcccggatcagtattacccgtgacacttctaagaaccatttttttctgcagctgaacagcgttaccgcagctgacactgcaacctactactgtgcccggggatattatggatacggagcttggttcgcttactggggccaaggcaccctcgtaactgtgagtgctgcttccaccaagggccc (SEQ ID NO: 193) muCD37-51gatgtgcagcttcaggagtcaggacctgacctgttgaaaccttctcagtcactacactcacctgcactgtcactggctactccatctccagtggttttgcctggcactggatccggcagtttccaggaaacaaactggaatggatgggctacatacactacagtggtagcactaactacagcccatctctcaaaagtcgaatctctatcactcgagactcatccaagaaccagttcttcctgcagttgaattctgtgactactgaggacacagccacatattactgtgcaagaggatactatggtttcggcgcctggtttgtttactggggccaagggactctggtcactgtctctgca (SEQ ID NO: 131) huCD37-51Aagcttgccaccatgggttggtcttgcatcatcctgttcctggtggccactgccactggcgtgcattcagaagttcagttggtggagtccggcccagaagtgctgaaacccggcgaatcactgtccctgacttgtaccgtgtcaggttatagcatcagcagcggctttgcttggcactggattcggcagtttccaggcaagggactggaatggatgggctacatccattacagtggctcaaccaattacagccctagcctgcagggccgaatctctattaccagggatagttctattaaccagtttttcctgcagcttaattccgtgactgcctctgacacagcaacttactattgcgcccgtggctactacgggttcggagcctggtttgtatactggggtcagggcaccctggtcactgtctcagccgcctctaccaagggccc (SEQ ID NO: 194) muCD37-56gatgtgcagcttcaggagtcaggacctgacctggtgaaaccttctcagtcactacactcacctgcactgtcactggctactccatcaccagtggttagcctggcactggatccggcagtaccaggaaacaaactggaatggatgggctacatacactacagtggtggcactaactacaacccatctctcaaaagtcgagtctctatcactcgagacacatccaagaaccagttcttcctgcagttgaattctgtgactactgaggacacagccacatattactgtgcaagaggctactatggMcggggcctggtagcttactggggccaagggactctggtccctgtctctgca (SEQ ID NO: 132) huCD37-56aagcttgccaccatggggtggagctgcattatcctgttcctcgtcgccaccgcaaccggcgtccactcccaggtgcagctgcaagaaagcgggccaggattggtaaaaccttcccagtctctgagtcttacttgtaccgtatctggatacagtatcacatctggcttcgcctggcattggattcgccagtacccggcaaggggcttgagtggatggggtatattcattattctggaggtaccaactacaacccttccctgaagagtcgagtctcaattaccagggacacttccaagaaccaattctttttgcagcttaattcagtgaccgctgccgacaccgctacttactactgcgcccggggctactatgggtttggtgcctggttcgcctactggggccaggggaccctggtgcccgtgtctgctgcctccacaaagggccc (SEQ ID NO: 133) muCD37-57gatgtgcagcttcaggagtcaggacctgacctgttgaaaccttctcagtcactacactcacctgcactgtcactggctactccatcaccagtggttagcctggcactggatccggcagtaccaggaaacaaactggaatggatgggctacatactctacagtggtagcactgtctacagcccatctctcaaaagtcgaatctctatcactcgagacacatccaagaaccagttcttcctgcagttgaattctgtgactactgaggacacagccacatattactgtgcaagagggtactatggttacggcgcctggtttgcttactggggccaagggactctggtcactgtctctgca (SEQ ID NO: 134)huCD37-57aagcttgccaccatgggctggagctgcatcattctgtttctggtggccacagcaactggcgttcacagtcaagtccaactgcaggagagcggccccggactcctgaaaccatctcagtcactcagtctgacatgtactgtgagcggctacagcattacctcaggcttcgcttggcattggatcaggcagttccccggaaaaggtctggagtggatggggtacattctgtacagcggcagtacagtgtattcaccctccttgaaatctaggatatcaatcacacgtgatacaagcaaaaatcagttcttcctccagctgaactccgtcaccgccgcagacacagcaacctattattgtgctcgcggatactacggatatggcgcatggttcgcctattggggccaggggacactcgtgaccgtttccgccgcctccacaaagggccc (SEQ ID NO: 135) 252-3gaggtgcaggtggtggagtctgggggagacttagtgaagcctggagggtccctgaaactctcctgtgcagcctctggattcactttcagtagctatggcatgtcttgggttcgccagactccagacaagaggctggagtgggtcgcaaccattagtagtggtggtagttacacctactctccagacagtgtgaaggggcgattcaccatctccagagacaatgccaagaaaaccctgtacctgcaaatgagcagtctgaagtctgaggacacagccatgtattactgtgcaagacatagttactacgatactagcgtcgactactggggtcaaggaacctcagtcaccgtctcctca (SEQ ID NO: 182)

TABLE 8  Variable light chain polynucleotide sequences AntibodyVL Polynucleotide Sequence (SEQ ID NO) muCD37-3gacatccagatgactcagtctccagcctccctactgtatctgtgggagaaactgtcaccatcacatgtcgagcaagtgagaatattcgcagtaatttagcatggtatcagcagaaacagggaaaatctcctcagctcctggtcaatgttgcaacaaacttagcagatggtgtgccatcaaggttcagtggcagtggatcaggcacacagtattccctcaagatcaacagcctgcagtctgaagattttgggacttattactgtcaacattattggggtactacgtggacgttcggtggaggcaccaagctggaaatcaaacgt (SEQ ID NO: 136) chCD37-3gaattcgccaccatgagtgtgcccactcaggtcctggggttgctgctgctgtggcttacagatgccagatgtgacatccagatgactcagtctccagcctccattctgtatctgtgggagaaactgtcaccatcacatgtcgagcaagtgagaatattcgcagtaatttagcatggtatcagcagaaacagggaaaatctcctcagctcctggtcaatgttgcaacaaacttagcagatggtgtgccatcaaggttcagtggcagtggatcaggcacacagtattccctcaagatcaacagcctgcagtctgaagattttgggacttattactgtcaacattattggggtactacgtggacgttcggtggaggcaccaagctggaaatcaaacgtacg (SEQ ID NO: 137) huCD37-3gaattcgccaccatgggttggtcctgcatcatcttgtactcgtggccacagccaccggtgttcactctgatatacaaatgac(1.0 and 1.1)tcaaagccottccagtttgagcgtaagtgtgggtgaacgcgtaacaatcacctgtagagctagtgaaaacatccgcagtaatctcgcatggtaccaacaaaagccaggtaagtcacctaagctcctcgtgaatgttgctaccaacctcgctgatggtgtgccttcacgattctctggttcaggttccggtaccgattattcacttaagatcaactcactccaaccagaagatttcggtacatattactgtcaacactactggggtacgacctggacattcggtcaaggtactaagctggaaatcaagcgtacg (SEQ ID NO: 138)muCD37-12gacattgtgctaacacagtctcctgcttccttagctgtatctctggggcagagggccaccatctcatgcagggccagccaaagtgtcagtacatctagctatagttatttgtactggttccagcagaaaccaggacagccacccaaactcctcatcaagtatgcatccaacctagcatctggggtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggaggaggatactgcaacatattactgtcaacacagttgggagattccgtacacgttcggaggggggaccaaactggaaataaaacgg (SEQ ID NO: 139)chCD37-12gaattcgccaccatgggttggtcctgtataatcctgttcttggtggccaccgctactggcgttcatagtgatattgtactcactcagtcaccagccagtctggcagtgtccctgggccagcgtgccaccatctcctgccgggcctcacagtccgtgagcactagctcttattcctatctctactggtttcaacagaagccaggacagccccctaagctgctgatcaagtacgcctccaacctcgccagcggcgttcccgctagattctctggttccggtagcggaactgatttcactttgaacatccaccccgttgaggaagaggataccgccacttactattgtcaacactcttgggagattccttacacctttggaggaggaacaaagctcgaaattaagcgtacg(SEQ ID NO: 140) muCD37-38caaattgttctcacccagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagtgccagctcaagtgtaacttacatgcactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaactggcttctggagtccctgctcgcttcagtggcggtgggtctgggacctcttactctctcacaatcagcagcatggaggctgaagatgctgccacttattactgccagcagtggattagtaacccacccacgttcggaggggggaccaagctggaaattaaacgg (SEQ ID NO: 141) chCD37-38gaattcgccaccatgggctggtcctgtatcatcctgtttctcgtggccacagctacaggtgttcattctcagattgtgctgacccaatcaccagctattatgtccgctagccccggcgagaaagtgacaatgacatgtagcgctagctcttctgtgacttacatgcattggtatcaacagaagtcaggtaccagtcccaagcgttggatctacgacacatccaaactggcctccggagtccctgccaggttcagcggaggtgggtccggcaccagttattcactgaccatatcctctatggaagctgaagatgctgctacttattattgtcaacaatggatttctaacccccccacctaggtggcggaacaaagctggagatcaagcgtacg (SEQ IDNO: 142) huCD37-38gaattcgccaccatgggatggtcctgcattattctgttcttggtcgccactgctactggcgttcactctgacattgtgctcacacagtctccagcctcaatgtctgcttcccccggtgagcgggtgaccatgacatgctctgccagttcctccgtgacatatatgcattggtatcagcaaaaacccggtacctctccaaaaagatggatctacgacacttcaaagcttgcatcaggcgttcctgccagattttccgggtctgggtctggcacttcatacagtctgaccattagttccatggaagctgaagatgcagccacctattactgtcagcagtggatttcaaatcctcctaccttcggcggcggaaccaaactggagataaagcgtacg (SEQ ID NO: 143)muCD37-50caaattgttctcacccagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagtgccacctcaagtgtgacttacatgcactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaactgccttatggagtccctggtcgtttcagtggtagtgggtctgggacctcttactctctcacaatcagcagcatggaggctgaagatgctgccacttattactgccagcagtggagtgataacccacccacgttcggctcggggacaaagttggaaataaagcgg (SEQ ID NO: 144) huCD37-50gaattcgccaccatgggttggtcatgcattattctgttcctggttgctaccgcaacaggagtacatagtgagatagtcctcacccaaagtcctgctactatgtctgccagcccaggagagcgtgtgaccatgacttgctctgcaacctcaagtgtgacatacatgcattggtatcagcaaaagcctggccaatcccctaaaaggtggatctacgatacttctaatctgccatacggtgtgcccgcaaggttctccgggagtggcagtggcaccagttatagtctgaccatcagttcaatggaagcagaggatgcagcaacctattattgtcagcagtggtccgataatccccctacttttggtcagggtacaaagctggagattaagcgtacg (SEQ ID NO: 145)muCD37-51caaattgttctcacccagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagtgccacctcaagtgtgacttacatgcactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaactggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcaacatggaggctgaagatgctgccacttattactgccagcagtggagtagtaacccacccacgttcggctcggggacaaagttggaaataaagcgg (SEQ ID NO: 146) huCD37-51gaattcgccaccatgggatggagctgtattattctgttcctggttgctactgctactggcgtccattccgagatagtcctcacccagagccccgcaaccatgagtgcctcccctggggagcgagtgactatgacttgttccgccacttcttcagttacctatatgcattggtatcagcagaaacctggacagtctccaaagcgttggatttacgacacctccaacctggcttcaggagttcctgctaggttcagcggatctgggtctggcacaagttattcactcaccattagttccatggaggccgaagatgccgctacttactactgtcagcagtggagcagcaacccccctacattcgggcagggaactaagctggagatcaaacgtacg (SEQ ID NO: 147)muCD37-56caaattgttctcacccagtctccagcattcatgtctgcatctccaggggataaggtcaccatgacctgcagtgccagttcaagtgttacttacatgcactggtatcagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaactggcttctggagtccctgctcgcttcagtggcggtgggtctgggacctcttactctctcacaatcagcaccatggaggctgaagatgctgccacttattactgccagcagtggattagtgacccacccacgttcggaggggggaccaagctggaaataaaacgg (SEQ ID NO: 148) huCD37-56gaattcgccaccatgggctggtcctgtatcatcctgtttctggtggcaaccgctactggggttcactctgatattgtcctgacacagagtccagccttcatgagtgcttctcccggagaaaaggtcacaatgacttgttcagcttcctcctccgtcacatacatgcattggtaccagcagaagcctgaccagagtcctaagaggtggatctatgatacaagcaatctggcttccggtgtcccctcccgcttttcaggcggcggaagcggaactgactatagccttaccatctcctcaatggaagccgaggacgctgctacatattactgccagcaatggatcagcgaccctcctacMcggacagggaacaaaattggaaattaagcgtacg (SEQ ID NO: 149)muCD37-57caaattgttctcacccagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagtgccacctcaagtgtgacttacatgcactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaactggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagcatggaggctgaagatgctgccacttattactgccagcagtggagtgataacccacccacgttcggctcggggacaaagttggaaataaagcgg (SEQ ID NO: 150) huCD37-57gaattcgccaccatggggtggtcctgtattatcctgttcctggtcgcaaccgccacaggcgttcactccgagatcgtgttgactcagagcccagccaccatgtccgcttcccccggggagagagtgacaatgacttgttccgccacaagttctgtaacctacatgcattggtaccagcaaaaaccaggacagagtccccgtcgttggatttatgatacctctaacctggcttcaggcgttcctgcccgcttttctggtagtggatctgggacttcctatagccttaccataagctctatggaagccgaggacgccgctacatactactgccagcagtggagtgataacccccccaccttcgggcagggaaccaaattggagatcaaacgtacg (SEQ ID NO: 151)252-3gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagcaattatttaaactggtatcagcagaaacccgatggaactgttaaactcctgatctactacacatcaaaattacactcaggagteccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatgcgcttccgtggacgttcggtggaggcaccaagctggaactcaaacgg (SEQ ID NO: 183)

TABLE 9  Full-length heavy chain polynucleotide sequences AntibodyFull-Length Heavy Chain Polynucleotide Sequence (SEQ ID NO) chCD37-3aagcttgccaccatggctgtcctggcactgctcctctgcctggtgacatacccaagctgtgtcctatcacaggtgcaggtgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccattacatgcactgtctcagggttctcattaaccacctctggtgtaagctgggttcgccagcctccaggaaagggtctggagtggctgggagtaatatggggtgacgggagcacaaactatcattcagctctcaaatccagactgagcatcaagaaggatcactccaagagccaagttttcttaaaactgaacagtctgcaaactgatgacacagccacgtactactgtgccaaaggaggctactcgttggctcactggggccaagggactctggtcacagtctctgcagcctctacgaagggcccatcagttttccccttggctccaagttctaaatccacaagcggtggaacagctgcactgggatgcctcgttaaagattatttccctgagcctgtgacagtgagctggaatagcggagcattgacttcaggtgtgcacacttttcccgctgtgttgcagtcctccggtctgtactcactgtccagtgtcgtaaccgtcccttctagcagcttgggaacccagacctacatctgtaacgtcaaccataaaccatccaacacaaaggtggataagaaggttgaaccaaagagctgtgataagacacatacatgccctccttgtcctgcaccagagctcctcggaggtccatctgtgttcctgtttccccccaaacccaaggacactcttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatcccgaggttaaattcaactggtacgtggatggagtcgaggttcacaatgccaagaccaagcccagggaggagcaatataattctacatatcgggtagtgagcgttctgaccgtgctccaccaagattggctcaatggaaaagagtacaagtgcaaggtgtccaacaaggctcttcccgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgcccccatctagagacgagctgaccaagaaccaggtgagtctcacttgtctggtcaaggggttttacccttctgacattgctgtagagtgggagtctaacggacagccagaaaacaactacaagacaactcccccagtgctggacagcgacgggagcttcttcctctactccaagttgactgtagacaagtctagatggcagcaaggaaacgttttctcctgctcagtaatgcatgaggctctgcacaatcactatacccagaaatcactgtcccttagcccagggtgactcgag (SEQ ID NO: 152) huCD37-3v1.0aagcttgccaccatgggttggagctgcattattctgtttctggtggccaccgccaccggtgtgcactcacaagtccaagtccaagaatctggtccaggtctggtggccccttcccaaactctgagcatcacctgtaccgtttctggttttagccttaccacctctggtgtgagttgggtacgccaaccacccggtaagggtctcgaatggctgggtgtaatctggggtgatggttccacaaattaccatccttccctcaagtcccgccttagcatcaaaaaggatcacagcaaaagtcaagttttcctgaaactgaatagtctgacagcagccgatacagccacctactattgcgccaagggtggttatagtcttgcacactggggtcaaggtaccctcgttaccgtctcctcagctagtaccaagggcccatcagttttccccttggctccaagttctaaatccacaagcggtggaacagctgcactgggatgcctcgttaaagattatttccctgagcctgtgacagtgagctggaatagcggagcattgacttcaggtgtgcacacttttcccgctgtgttgcagtcctccggtctgtactcactgtccagtgtcgtaaccgtcccttctagcagcttgggaacccagacctacatctgtaacgtcaaccataaaccatccaacacaaaggtggataagaaggttgaaccaaagagctgtgataagacacatacatgccctccttgtcctgcaccagagctcctcggaggtccatctgtgttcctgtttccccccaaacccaaggacactcttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatcccgaggttaaattcaactggtacgtggatggagtcgaggttcacaatgccaagaccaagcccagggaggagcaatataattctacatatcgggtagtgagcgttctgaccgtgctccaccaagattggctcaatggaaaagagtacaagtgcaaggtgtccaacaaggctcttcccgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgcccccatctagagacgagctgaccaagaaccaggtgagtctcacttgtctggtcaaggggttttacccttctgacattgctgtagagtgggagtctaacggacagccagaaaacaactacaagacaactcccccagtgctggacagcgacgggagcttcttcctctactccaagttgactgtagacaagtctagatggcagcaaggaaacgttttctcctgctcagtaatgcatgaggctctgcacaatcactatacccagaaatcactgtcccttagcccagggtgactcgag (SEQ ID NO: 153) huCD37-3v1.1aagcttgccaccatgggctggagctgtatcattctgtactggtggcgacagctactggggtccactcccaagtgcaggtacaagagtccgggcctggattggtcgcaccaagccagaccctctctatcacttgtaccgttagcgggttctctctgacaaccagtggagtgagttgggtgaggcagccaccaggaaagggactggagtggctgggggtgataggggcgacggcagcacaaactatcattccagtcttaaatctcggttgtccattaaaaaagaccatagtaaatctcaagttttcctgaaactcaatagcctgacagccgcagacactgctacgtattactgcgccaaaggaggatacagtctggctcactggggacaggggaccctggtgaccgtgtcatccgcatcaacaaagggcccatcagttttccccttggctccaagttctaaatccacaagcggtggaacagctgcactgggatgcctcgttaaagattatttccctgagcctgtgacagtgagctggaatagcggagcattgacttcaggtgtgcacacttttcccgctgtgttgcagtcctccggtctgtactcactgtccagtgtcgtaaccgtcccttctagcagcttgggaacccagacctacatctgtaacgtcaaccataaaccatccaacacaaaggtggataagaaggttgaaccaaagagctgtgataagacacatacatgccctccttgtcctgcaccagagctcctcggaggtccatctgtgttcctgtttccccccaaacccaaggacactcttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatcccgaggttaaattcaactggtacgtggatggagtcgaggttcacaatgccaagaccaagcccagggaggagcaatataattctacatatcgggtagtgagcgttctgaccgtgctccaccaagattggctcaatggaaaagagtacaagtgcaaggtgtccaacaaggctcttcccgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgcccccatctagagacgagctgaccaagaaccaggtgagtctcacttgtctggtcaaggggttttacccttctgacattgctgtagagtgggagtctaacggacagccagaaaacaactacaagacaactcccccagtgctggacagcgacgggagcttcttcctctactccaagttgactgtagacaagtctagatggcagcaaggaaacgttttctcctgctcagtaatgcatgaggctctgcacaatcactatacccagaaatcactgtcccttagcccagggtgactcgag (SEQ ID NO: 154) chCD37-12aagcttgccaccatggggtggtcatgcataatcctctactggtcgctactgctaccggtgtgcactcacagattcagctggttcaaagtggcccagagctgaaaaagccaggggaaacagtgaaaataagttgcaaggcatccggttacactacacaaagtacggcatgaactgggtcaagcaggcccagggcaaggggctcaaatggatgggttggatcaataccaacactggcgagtctaggaatgctgaggagtttaagggccggtttgccttcagcctggagacaagtgccagcacagcttacctgcaaatcaacaatctgaagtatgaggatacagcaacctatactgcggccgcggcactgtcgttgcagactggggacaaggtaccaccttgactgtatccagtgccagcactaagggcccatcagtatccccttggctccaagttctaaatccacaagcggtggaacagctgcactgggatgcctcgttaaagattatttccctgagcctgtgacagtgagctggaatagcggagcattgacttcaggtgtgcacacttttcccgctgtgttgcagtcctccggtctgtactcactgtccagtgtcgtaaccgtcccttctagcagcttgggaacccagacctacatctgtaacgtcaaccataaaccatccaacacaaaggtggataagaaggttgaaccaaagagctgtgataagacacatacatgccctccttgtcctgcaccagagctcctcggaggtccatctgtgttcctgtttccccccaaacccaaggacactcttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatcccgaggttaaattcaactggtacgtggatggagtcgaggttcacaatgccaagaccaagcccagggaggagcaatataattctacatatcgggtagtgagcgttctgaccgtgctccaccaagattggctcaatggaaaagagtacaagtgcaaggtgtccaacaaggctcttcccgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgcccccatctagagacgagctgaccaagaaccaggtgagtctcacttgtctggtcaaggggttttacccttctgacattgctgtagagtgggagtctaacggacagccagaaaacaactacaagacaactcccccagtgctggacagcgacgggagcttcttcctctactccaagttgactgtagacaagtctagatggcagcaaggaaacgttttctcctgctcagtaatgcatgaggctctgcacaatcactatacccagaaatcactgtcccttagcccagggtgactcgag (SEQ ID NO: 155) chCD37-38aagcttgccaccatgggctggagttgtatcattctgtttttggtggccaccgccactggagtccattcccaagtgcaactccaggaatctggccctgacctggttaagccatctcagagcctctccctgacctgcactgttacaggatactcaatcacatcaggctttggctggcactggatcagacaatttcccgggaacaagttggaatggatggcttacattctgtatagcgggggtaccgattacaatccttccctcaagagccgaatctctatcaccagggatacaagcaagaaccaattttttctccgcctcagctctgtgactaccgaagataccgctacttactattgtgccaggggctactatggatatggtgcatggttcgtctattggggccagggaaccctggtgactgtgagcgctgcctctaccaagggcccatcagttttccccttggctccaagttctaaatccacaagcggtggaacagctgcactgggatgcctcgttaaagattatttccctgagcctgtgacagtgagctggaatagcggagcattgacttcaggtgtgcacacttttcccgctgtgttgcagtcctccggtctgtactcactgtccagtgtcgtaaccgtcccttctagcagcttgggaacccagacctacatctgtaacgtcaaccataaaccatccaacacaaaggtggataagaaggttgaaccaaagagctgtgataagacacatacatgccctccttgtcctgcaccagagctcctcggaggtccatctgtgttcctgtttccccccaaacccaaggacactcttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatcccgaggttaaattcaactggtacgtggatggagtcgaggttcacaatgccaagaccaagcccagggaggagcaatataattctacatatcgggtagtgagcgttctgaccgtgctccaccaagattggctcaatggaaaagagtacaagtgcaaggtgtccaacaaggctcttcccgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgcccccatctagagacgagctgaccaagaaccaggtgagtctcacttgtctggtcaaggggttttacccttctgacattgctgtagagtgggagtctaacggacagccagaaaacaactacaagacaactcccccagtgctggacagcgacgggagcttcttcctctactccaagttgactgtagacaagtctagatggcagcaaggaaacgttttctcctgctcagtaatgcatgaggctctgcacaatcactatacccagaaatcactgtcccttagcccagggtgactcgag (SEQ ID NO: 156)huCD37-38aagcttgccaccatgggttggagctgcatcattcttttcctggtcgctactgcaactggagtccactcacaggtccagctgcaagagtccggtcctgggcttgtgaaacccagccagtccctcagtctcacctgtactgtctctggctactctattaccagtgggttcggctggcattggattaggcagtttcccggtaaggggctggagtggatggcatatatcctgtacagcggaggaaccgattacaacccaagtctgaagagcaggatcagcattacccgggacacaagcaaaaaccagtttttccttcggctgtctagtgttacagctgcagacaccgctacttactattgtgctcggggttactatggctatggggcttggtttgtgtattggggacaaggcactcttgtgaccgtgagcagcgcctcaacaaagggcccatcagttttccccttggctccaagttctaaatccacaagcggtggaacagctgcactgggatgcctcgttaaagattatttccctgagcctgtgacagtgagctggaatagcggagcattgacttcaggtgtgcacacttttcccgctgtgttgcagtcctccggtctgtactcactgtccagtgtcgtaaccgtcccttctagcagcttgggaacccagacctacatctgtaacgtcaaccataaaccatccaacacaaaggtggataagaaggttgaaccaaagagctgtgataagacacatacatgccctccttgtcctgcaccagagctcctcggaggtccatctgtgttcctgtttccccccaaacccaaggacactcttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatcccgaggttaaattcaactggtacgtggatggagtcgaggttcacaatgccaagaccaagcccagggaggagcaatataattctacatatcgggtagtgagcgttctgaccgtgctccaccaagattggctcaatggaaaagagtacaagtgcaaggtgtccaacaaggctcttcccgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgcccccatctagagacgagctgaccaagaaccaggtgagtctcacttgtctggtcaaggggttttacccttctgacattgctgtagagtgggagtctaacggacagccagaaaacaactacaagacaactcccccagtgctggacagcgacgggagcttcttcctctactccaagttgactgtagacaagtctagatggcagcaaggaaacgttttctcctgctcagtaatgcatgaggctctgcacaatcactatacccagaaatcactgtcccttagcccagggtgactcgag (SEQ ID NO: 157)huCD37-50aagcttgccaccatggggtggtcctgcataatccttttcctggttgctactgctaccggagtccattcacaggtgcagctgcaggagtccggccccggcctgctcaagccttctcagagtctgagtctgacttgtactgtttctggctacagcataaccagcggtttcgcttggcactggatcagacagcatcccggcaacaaactggagtggatgggatacatactgtactcaggctcaactgtctattccccctccctgaaatcccggatcagtattacccgtgacacttctaagaaccatttttttctgcagctgaacagcgttaccgcagctgacactgcaacctactactgtgcccggggatattatggatacggagcttggttcgcttactggggccaaggcaccctcgtaactgtgagtgctgcttccaccaagggcccatcagttttccccttggctccaagttctaaatccacaagcggtggaacagctgcactgggatgcctcgttaaagattatttccctgagcctgtgacagtgagctggaatagcggagcattgacttcaggtgtgcacacttttcccgctgtgttgcagtcctccggtctgtactcactgtccagtgtcgtaaccgtcccttctagcagcttgggaacccagacctacatctgtaacgtcaaccataaaccatccaacacaaaggtggataagaaggttgaaccaaagagctgtgataagacacatacatgccctccttgtcctgcaccagagctcctcggaggtccatctgtgttcctgtttccccccaaacccaaggacactcttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatcccgaggttaaattcaactggtacgtggatggagtcgaggttcacaatgccaagaccaagcccagggaggagcaatataattctacatatcgggtagtgagcgttctgaccgtgctccaccaagattggctcaatggaaaagagtacaagtgcaaggtgtccaacaaggctcttcccgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgcccccatctagagacgagctgaccaagaaccaggtgagtctcacttgtctggtcaaggggttttacccttctgacattgctgtagagtgggagtctaacggacagccagaaaacaactacaagacaactcccccagtgctggacagcgacgggagcttcttcctctactccaagttgactgtagacaagtctagatggcagcaaggaaacgttttctcctgctcagtaatgcatgaggctctgcacaatcactatacccagaaatcactgtcccttagcccagggtgactcgag (SEQ ID NO: 158)huCD37-51aagcttgccaccatgggttggtcttgcatcatcctgttcctggtggccactgccactggcgtgcattcagaagttcagttggtggagtccggcccagaagtgctgaaacccggcgaatcactgtccctgacttgtaccgtgtcaggttatagcatcagcagcggctagcttggcactggattcggcagtaccaggcaagggactggaatggatgggctacatccattacagtggctcaaccaattacagccctagcctgcagggccgaatctctattaccagggatagttctattaaccagtttttcctgcagcttaattccgtgactgcctctgacacagcaacttactattgcgcccgtggctactacgggttcggagcctggtttgtatactggggtcagggcaccctggtcactgtctcagccgcctctaccaagggcccatcagttttccccttggctccaagttctaaatccacaagcggtggaacagctgcactgggatgcctcgttaaagattatttccctgagcctgtgacagtgagctggaatagcggagcattgacttcaggtgtgcacacttttcccgctgtgttgcagtcctccggtctgtactcactgtccagtgtcgtaaccgtcccttctagcagcttgggaacccagacctacatctgtaacgtcaaccataaaccatccaacacaaaggtggataagaaggttgaaccaaagagctgtgataagacacatacatgccctccttgtcctgcaccagagctcctcggaggtccatctgtgttcctgtttccccccaaacccaaggacactcttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatcccgaggttaaattcaactggtacgtggatggagtcgaggttcacaatgccaagaccaagcccagggaggagcaatataattctacatatcgggtagtgagcgttctgaccgtgctccaccaagattggctcaatggaaaagagtacaagtgcaaggtgtccaacaaggctcttcccgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgcccccatctagagacgagctgaccaagaaccaggtgagtctcacttgtctggtcaaggggttttacccttctgacattgctgtagagtgggagtctaacggacagccagaaaacaactacaagacaactcccccagtgctggacagcgacgggagcttcttcctctactccaagttgactgtagacaagtctagatggcagcaaggaaacgttttctcctgctcagtaatgcatgaggctctgcacaatcactatacccagaaatcactgtcccttagcccagggtgactcgag (SEQ ID NO: 159)huCD37-56aagcttgccaccatggggtggagctgcattatcctgttcctcgtcgccaccgcaaccggcgtccactcccaggtgcagctgcaagaaagcgggccaggattggtaaaaccttcccagtctctgagtcttacttgtaccgtatctggatacagtatcacatctggcttcgcctggcattggattcgccagtacccggcaaggggcttgagtggatggggtatattcattattctggaggtaccaactacaacccttccctgaagagtcgagtctcaattaccagggacacttccaagaaccaattctttttgcagcttaattcagtgaccgctgccgacaccgctacttactactgcgcccggggctactatgggtttggtgcctggttcgcctactggggccaggggaccctggtgcccgtgtctgctgcctccacaaagggcccatcagttttccccttggctccaagttctaaatccacaagcggtggaacagctgcactgggatgcctcgttaaagattatttccctgagcctgtgacagtgagctggaatagcggagcattgacttcaggtgtgcacacttttcccgctgtgttgcagtcctccggtctgtactcactgtccagtgtcgtaaccgtcccttctagcagcttgggaacccagacctacatctgtaacgtcaaccataaaccatccaacacaaaggtggataagaaggttgaaccaaagagctgtgataagacacatacatgccctccttgtcctgcaccagagctcctcggaggtccatctgtgttcctgtttccccccaaacccaaggacactcttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatcccgaggttaaattcaactggtacgtggatggagtcgaggttcacaatgccaagaccaagcccagggaggagcaatataattctacatatcgggtagtgagcgttctgaccgtgctccaccaagattggctcaatggaaaagagtacaagtgcaaggtgtccaacaaggctcttcccgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgcccccatctagagacgagctgaccaagaaccaggtgagtctcacttgtctggtcaaggggttttacccttctgacattgctgtagagtgggagtctaacggacagccagaaaacaactacaagacaactcccccagtgctggacagcgacgggagcttcttcctctactccaagttgactgtagacaagtctagatggcagcaaggaaacgttttctcctgctcagtaatgcatgaggctctgcacaatcactatacccagaaatcactgtcccttagcccagggtgactcgag (SEQ ID NO: 160)huCD37-57aagcttgccaccatgggctggagctgcatcattctgtttctggtggccacagcaactggcgttcacagtcaagtccaactgcaggagagcggccccggactcctgaaaccatctcagtcactcagtctgacatgtactgtgagcggctacagcattacctcaggcttcgcttggcattggatcaggcagttccccggaaaaggtctggagtggatggggtacattctgtacagcggcagtacagtgtattcaccctccttgaaatctaggatatcaatcacacgtgatacaagcaaaaatcagttcttcctccagctgaactccgtcaccgccgcagacacagcaacctattattgtgctcgcggatactacggatatggcgcatggttcgcctattggggccaggggacactcgtgaccgtttccgccgcctccacaaagggcccatcagttaccccttggctccaagttctaaatccacaagcggtggaacagctgcactgggatgcctcgttaaagattatttccctgagcctgtgacagtgagctggaatagcggagcattgacttcaggtgtgcacacttttcccgctgtgttgcagtcctccggtctgtactcactgtccagtgtcgtaaccgtcccttctagcagcttgggaacccagacctacatctgtaacgtcaaccataaaccatccaacacaaaggtggataagaaggttgaaccaaagagctgtgataagacacatacatgccctccttgtcctgcaccagagctcctcggaggtccatctgtgttcctgtttccccccaaacccaaggacactcttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatcccgaggttaaattcaactggtacgtggatggagtcgaggttcacaatgccaagaccaagcccagggaggagcaatataattctacatatcgggtagtgagcgttctgaccgtgctccaccaagattggctcaatggaaaagagtacaagtgcaaggtgtccaacaaggctcttcccgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgcccccatctagagacgagctgaccaagaaccaggtgagtctcacttgtctggtcaaggggttttacccttctgacattgctgtagagtgggagtctaacggacagccagaaaacaactacaagacaactcccccagtgctggacagcgacgggagcttcttcctctactccaagttgactgtagacaagtctagatggcagcaaggaaacgttttctcctgctcagtaatgcatgaggctctgcacaatcactatacccagaaatcactgtcccttagcccagggtgactcgag (SEQ ID NO: 161)

TABLE 10  Full-length light chain polynucleotide sequences AntibodyFull-length Light Chain Polynucleotide Sequence (SEQ ID NO) chCD37-3gaattcgccaccatgagtgtgcccactcaggtcctggggttgctgctgctgtggcttacagatgccagatgtgacatccagatgactcagtctccagcctccctttctgtatctgtgggagaaactgtcaccatcacatgtcgagcaagtgagaatattcgcagtaatttagcatggtatcagcagaaacagggaaaatctcctcagctcctggtcaatgttgcaacaaacttagcagatggtgtgccatcaaggttcagtggcagtggatcaggcacacagtattccctcaagatcaacagcctgcagtctgaagattttgggacttattactgtcaacattattggggtactacgtggacgttcggtggaggcaccaagctggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag(SEQ ID NO: 162) huCD37-3gaattcgccaccatgggttggtcctgcatcatcttgtactcgtggccacagccaccggtgttcactctgatatacaaatgac(1.0 and 1.1)tcaaagcccttccagtttgagcgtaagtgtgggtgaacgcgtaacaatcacctgtagagctagtgaaaacatccgcagtaatctcgcatggtaccaacaaaagccaggtaagtcacctaagctcctcgtgaatgttgctaccaacctcgctgatggtgtgccttcacgattctctggttcaggttccggtaccgattattcacttaagatcaactcactccaaccagaagatttcggtacatattactgtcaacactactggggtacgacctggacattcggtcaaggtactaagctggaaatcaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag (SEQ ID NO: 163)chCD37-12gaattcgccaccatgggttggtcctgtataatcctgttcttggtggccaccgctactggcgttcatagtgatattgtactcactcagtcaccagccagtctggcagtgtccctgggccagcgtgccaccatctcctgccgggcctcacagtccgtgagcactagctcttattcctatctctactggtacaacagaagccaggacagccccctaagctgctgatcaagtacgcctccaacctcgccagcggcgttcccgctagattctctggttccggtagcggaactgatttcactttgaacatccaccccgttgaggaagaggataccgccacttactattgtcaacactcttgggagattccttacacctaggaggaggaacaaagctcgaaattaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag (SEQ ID NO: 164) chCD37-38gaattcgccaccatgggctggtcctgtatcatcctgtttctcgtggccacagctacaggtgttcattctcagattgtgctgacccaatcaccagctattatgtccgctagccccggcgagaaagtgacaatgacatgtagcgctagctcttctgtgacttacatgcattggtatcaacagaagtcaggtaccagtcccaagcgttggatctacgacacatccaaactggcctccggagtccctgccaggttcagcggaggtgggtccggcaccagttattcactgaccatatcctctatggaagctgaagatgctgctacttattattgtcaacaatggatactaacccccccacctaggtggcggaacaaagctggagatcaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag (SEQ ID NO: 165)huCD37-38gaattcgccaccatgggatggtcctgcattattctgttcttggtcgccactgctactggcgttcactctgacattgtgctcacacagtctccagcctcaatgtctgcttcccccggtgagcgggtgaccatgacatgctctgccagttcctccgtgacatatatgcattggtatcagcaaaaacccggtacctctccaaaaagatggatctacgacacttcaaagcttgcatcaggcgttcctgccagattttccgggtctgggtctggcacttcatacagtctgaccattagttccatggaagctgaagatgcagccacctattactgtcagcagtggatttcaaatcctcctaccttcggcggcggaaccaaactggagataaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag (SEQ ID NO: 166)huCD37-50gaattcgccaccatgggttggtcatgcattattctgttcctggttgctaccgcaacaggagtacatagtgagatagtcctcacccaaagtcctgctactatgtctgccagcccaggagagcgtgtgaccatgacttgctctgcaacctcaagtgtgacatacatgcattggtatcagcaaaagcctggccaatcccctaaaaggtggatctacgatacttctaatctgccatacggtgtgcccgcaaggttctccgggagtggcagtggcaccagttatagtctgaccatcagttcaatggaagcagaggatgcagcaacctattattgtcagcagtggtccgataatccccctacttttggtcagggtacaaagctggagattaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag (SEQ ID NO: 167)huCD37-51gaattcgccaccatgggatggagctgtattattctgttcctggttgctactgctactggcgtccattccgagatagtcctcacccagagccccgcaaccatgagtgcctcccctggggagcgagtgactatgacttgttccgccacttcttcagttacctatatgcattggtatcagcagaaacctggacagtctccaaagcgttggatttacgacacctccaacctggcttcaggagttcctgctaggttcagcggatctgggtctggcacaagttattcactcaccattagttccatggaggccgaagatgccgctacttactactgtcagcagtggagcagcaacccccctacattcgggcagggaactaagctggagatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag (SEQ ID NO: 168)huCD37-56gaattcgccaccatgggctggtcctgtatcatcctgtttctggtggcaaccgctactggggttcactctgatattgtcctgacacagagtccagccttcatgagtgcttctcccggagaaaaggtcacaatgacttgttcagcttcctcctccgtcacatacatgcattggtaccagcagaagcctgaccagagtcctaagaggtggatctatgatacaagcaatctggcttccggtgtcccctcccgcttttcaggcggcggaagcggaactgactatagccttaccatctcctcaatggaagccgaggacgctgctacatattactgccagcaatggatcagcgaccctcctactttcggacagggaacaaaattggaaattaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag (SEQ ID NO: 169)huCD37-57gaattcgccaccatggggtggtcctgtattatcctgttcctggtcgcaaccgccacaggcgttcactccgagatcgtgttgactcagagcccagccaccatgtccgcttcccccggggagagagtgacaatgacttgttccgccacaagttctgtaacctacatgcattggtaccagcaaaaaccaggacagagtccccgtcgttggatttatgatacctctaacctggcttcaggcgttcctgcccgcttttctggtagtggatctgggacttcctatagccttaccataagctctatggaagccgaggacgccgctacatactactgccagcagtggagtgataacccccccaccttcgggcagggaaccaaattggagatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag (SEQ ID NO: 170)

Also provided is a polynucleotide having at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about 99%sequence identity to SEQ ID NOs:121-170, 182, or 183. Thus, in certainembodiments, the polynucleotide comprises (a) a polynucleotide having atleast about 95% sequence identity to SEQ ID NOs:121-135, 152-161, or182, and/or (b) a polynucleotide having at least about 95% sequenceidentity to SEQ ID NOs:136-151, 162-170, or 183. In certain embodiments,the polynucleotide comprises (a) a polynucleotide having the nucleicacid sequence of SEQ ID NOs: 121-135, 152-161 or 182; and/or (b) apolynucleotide having the nucleic acid sequence of SEQ ID NOs: 136-151,162-170, or 183.

In some embodiments, the polynucleotide encodes the light chain encodedby the recombinant plasmid DNA phuCD37-3LC (ATCC Deposit DesignationPTA-10722, deposited with the ATCC on Mar. 18, 2010) or a light chainthat is at least about 85%, at least about 90%, at least about 95%, orat least about 99% to the light chain encoded by phuCD37-3LC(PTA-10722). In some embodiments, the polynucleotide encodes the heavychain encoded by the recombinant plasmid DNA phuCD37-3HCv.1.0 (ATCCDeposit Designation PTA-10723, deposited with the ATCC on Mar. 18, 2010)or a heavy chain that is at least about 85%, at least about 90%, atleast about 95%, or at least about 99% identical to the heavy chainencoded by phuCD37-3HCv.1.0 (PTA-10723). In certain embodiments thepolynucleotide is the recombinant plasmid DNA phuCD37-3LC (PTA-10722) orthe recombinant plasmid phuCD37-3HCv.1.0 (PTA-10723).

In certain embodiments the polynucleotides comprise the coding sequencefor the mature polypeptide fused in the same reading frame to apolynucleotide which aids, for example, in expression and secretion of apolypeptide from a host cell (e.g. a leader sequence which functions asa secretory sequence for controlling transport of a polypeptide from thecell). The polypeptide having a leader sequence is a preprotein and canhave the leader sequence cleaved by the host cell to form the matureform of the polypeptide. The polynucleotides can also encode for aproprotein which is the mature protein plus additional 5′ amino acidresidues. A mature protein having a prosequence is a proprotein and isan inactive form of the protein. Once the prosequence is cleaved anactive mature protein remains.

In certain embodiments the polynucleotides comprise the coding sequencefor the mature polypeptide fused in the same reading frame to a markersequence that allows, for example, for purification of the encodedpolypeptide. For example, the marker sequence can be a hexa-histidinetag supplied by a pQE-9 vector to provide for purification of the maturepolypeptide fused to the marker in the case of a bacterial host, or themarker sequence can be a hemagglutinin (HA) tag derived from theinfluenza hemagglutinin protein when a mammalian host (e.g. COS-7 cells)is used.

The present invention further relates to variants of the hereinabovedescribed polynucleotides encoding, for example, fragments, analogs, andderivatives.

The polynucleotide variants can contain alterations in the codingregions, non-coding regions, or both. In some embodiments thepolynucleotide variants contain alterations which produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded polypeptide. In some embodiments,nucleotide variants are produced by silent substitutions due to thedegeneracy of the genetic code. Polynucleotide variants can be producedfor a variety of reasons, e.g., to optimize codon expression for aparticular host (change codons in the human mRNA to those preferred by abacterial host such as E. coli).

Vectors and cells comprising the polynucleotides described herein arealso provided.

IV. Methods of Use and Pharmaceutical Compositions

The CD37-binding agents (including antibodies, immunoconjugates, andpolypeptides) of the invention are useful in a variety of applicationsincluding, but not limited to, therapeutic treatment methods, such asthe treatment of cancer, such as B-cell malignancies, autoimmunediseases, and inflammatory diseases. In certain embodiments, the agentsare useful for depleting B-cells. In certain embodiments, the agents areuseful for depleting autoreactive B-cells. In certain embodiments, theagents are useful for depleting peripheral B-cells. In certainembodiments, the agents are useful for preventing inappropriate T-cellstimulation. The T-cell stimulation can be in connection with a B-cellpathway. The methods of use can be in vitro, ex vivo, or in vivomethods. In certain embodiments, the CD37-binding agent or antibody orimmunoconjugate, or polypeptide is an antagonist of the human CD37 towhich it binds.

In one aspect, anti-CD37 antibodies and immunoconjugates of theinvention are useful for detecting the presence of CD37 in a biologicalsample. The term “detecting” as used herein encompasses quantitative orqualitative detection. In certain embodiments, a biological samplecomprises a cell or tissue. In certain embodiments, such tissues includetissues that express CD37 at higher levels relative to other tissues,for example, B-cells and/or B-cell associated tissues.

In one aspect, the invention provides a method of detecting the presenceof CD37 in a biological sample. In certain embodiments, the methodcomprises contacting the biological sample with an anti-CD37 antibodyunder conditions permissive for binding of the anti-CD37 antibody toCD37, and detecting whether a complex is formed between the anti-CD37antibody and CD37.

In one aspect, the invention provides a method of diagnosing a disorderassociated with increased expression of CD37. In certain embodiments,the method comprises contacting a test cell with an anti-CD37 antibody;determining the level of expression (either quantitatively orqualitatively) of CD37 by the test cell by detecting binding of theanti-CD37 antibody to CD37; and comparing the level of expression ofCD37 by the test cell with the level of expression of CD37 by a controlcell (e.g., a normal cell of the same tissue origin as the test cell ora cell that expresses CD37 at levels comparable to such a normal cell),wherein a higher level of expression of CD37 by the test cell ascompared to the control cell indicates the presence of a disorderassociated with increased expression of CD37. In certain embodiments,the test cell is obtained from an individual suspected of having anautoimmune disorder or inflammatory disorder. In some embodiments, thedisorder is associated with increased expression of CD37. In someembodiments, the disorder is associated with increased number ofB-cells. In some embodiments, the disorder is associated with increasedactivity of B-cells.

In certain embodiments, a method of diagnosis or detection, such asthose described above, comprises detecting binding of an anti-CD37antibody to CD37 expressed on the surface of a cell or in a membranepreparation obtained from a cell expressing CD37 on its surface. Incertain embodiments, the method comprises contacting a cell with ananti-CD37 antibody under conditions permissive for binding of theanti-CD37 antibody to CD37, and detecting whether a complex is formedbetween the anti-CD37 antibody and CD37 on the cell surface. Anexemplary assay for detecting binding of an anti-CD37 antibody to CD37expressed on the surface of a cell is a “FACS” assay.

Certain other methods can be used to detect binding of anti-CD37antibodies to CD37. Such methods include, but are not limited to,antigen-binding assays that are well known in the art, such as westernblots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay),“sandwich” immunoassays, immunoprecipitation assays, fluorescentimmunoassays, protein A immunoassays, and immunohistochemistry (IHC).

In certain embodiments, anti-CD37 antibodies are labeled. Labelsinclude, but are not limited to, labels or moieties that are detecteddirectly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction.

In certain embodiments, anti-CD37 antibodies are immobilized on aninsoluble matrix. Immobilization entails separating the anti-CD37antibody from any CD37 that remains free in solution. Thisconventionally is accomplished by either insolubilizing the anti-CD37antibody before the assay procedure, as by adsorption to awater-insoluble matrix or surface (Bennich et al., U.S. Pat. No.3,720,760), or by covalent coupling (for example, using glutaraldehydecross-linking), or by insolubilizing the anti-CD37 antibody afterformation of a complex between the anti-CD37 antibody and CD37, e.g., byimmunoprecipitation.

Any of the above embodiments of diagnosis or detection can be carriedout using an immunoconjugate of the invention in place of or in additionto an anti-CD37 antibody.

In certain embodiments, the disease treated with the CD37-binding agentis an autoimmune or inflammatory disease. In certain embodiments, theautoimmune or inflammatory disease is selected from the group consistingof psoriasis, dermatitis, systemic scleroderma and sclerosis, responsesassociated with inflammatory bowel disease, Crohn's disease, ulcerativecolitis, respiratory distress syndrome, adult respiratory distresssyndrome (ARDS), dermatitis, meningitis, encephalitis, uveitis, colitis,glomerulonephritis, allergic conditions, eczema, asthma, conditionsinvolving infiltration of T cells and chronic inflammatory responses,atherosclerosis, leukocyte adhesion deficiency, rheumatoid arthritis,systemic lupus erythematosus (SLE), diabetes mellitus, multiplesclerosis, Reynaud's syndrome, autoimmune thyroiditis, allergicencephalomyelitis, Sjorgen's syndrome, juvenile onset diabetes, immuneresponses associated with acute and delayed hypersensitivity mediated bycytokines and T-lymphocytes, tuberculosis, sarcoidosis, polymyositis,granulomatosis, vasculitis, pernicious anemia (Addison's disease),diseases involving leukocyte diapedesis, central nervous system (CNS)inflammatory disorder, multiple organ injury syndrome, hemolytic anemia,myasthenia gravis, antigen-antibody complex mediated diseases,anti-glomerular basement membrane disease, antiphospholipid syndrome,allergic neuritis, Graves' disease, Lambert-Eaton myasthenic syndrome,pemphigoid bullous, pemphigus, autoimmune polyendocrinopathies, Reiter'sdisease, stiff-man syndrome, Behcet disease, giant cell arteritis,immune complex nephritis, IgA nephropathy, IgM polyneuropathies,idiopathic thrombocytopenic purpura (ITP) and autoimmunethrombocytopenia.

In some embodiments, the autoimmune or inflammatory disease is selectedfrom the group consisting of: RA, lupus, immune thrombocytopenicpurpura, pure red cell aplasia, autoimmune anemia, cold agglutinindisease, type B syndrome of severe insulin resistance, mixedcryoglobulinermia, myasthenia gravis, Wegener's granulomatosis,microscopic polyangiitis (MPA), refractory pemphigus vulgaris,dermatomyositis, Sjogren's syndrome, active type-II mixedcryoglobulinemia, pemphigus vulgaris, autoimmune neuropathy,paraneoplastic opsoclonus-myoclonus syndrome, and relapsing-remittingmultiple sclerosis (RRMS).

In certain embodiments, the autoimmune disease or inflammatory diseaseis characterized by CD37 expressing cells to which the CD37-bindingagent (e.g., antibody) binds.

The present invention provides for methods of treating autoimmune andinflammatory diseases comprising administering a therapeuticallyeffective amount of a CD37-binding agent to a subject (e.g., a subjectin need of treatment). In certain embodiments, the subject is a human.

The present invention further provides methods for depleting B-cells,e.g., autoreactive B-cells, using the antibodies or other agentsdescribed herein. In certain embodiments, the method of depletingB-cells comprises contacting a B-cell with a CD37-binding agent (e.g.,antibody) in vitro. For example, a cell line that expresses CD37 iscultured in medium to which is added the antibody or other agent todeplete the cells. In some embodiments, the cells are isolated from apatient sample such as, for example, a tissue biopsy, pleural effusion,or blood sample and cultured in medium to which is added an CD37-bindingagent to deplete the cells.

In some embodiments, the method of depleting B-cells, e.g. autoreactiveB-cells, comprises contacting the cells with the CD37-binding agent(e.g., antibody) in vivo. In certain embodiments, contacting a cell witha CD37-binding agent is undertaken in an animal model. For example,CD37-binding agents can be administered to xenografts expressing one ormore CD37s that have been grown in immunocompromised mice (e.g. NOD/SCIDmice). In some embodiments, cells are isolated from a patient samplesuch as, for example, a tissue biopsy, pleural effusion, or blood sampleand injected into immunocompromised mice that are then administered aCD37-binding agent to deplete B-cells. In some embodiments, theCD37-binding agent is administered at the same time or shortly afterintroduction of cells into the animal. In further examples, CD37 bindingagents can be administered in vivo to mice expressing one or more CD37antigens. In some embodiments, these mice can be engineered to expresshuman CD37 in addition to, or instead of, murine CD37. In someembodiments, these mice are disease models, e.g. models for autoimmunedisease. In some embodiments, administering a CD37 binding agentdepletes B-cells in vivo. In some embodiments, a CD37 binding agentprevents T-cell stimulation. In some embodiments, administering a CD37binding agent prevents or alleviates an autoimmune disease.

In certain embodiments, the B-cells overexpress CD37. In otherembodiments, the B-cells do not overexpress CD37. In some embodiments,the B-cells are not cancer cells. In some embodiments, the B-cells arenot tumor cells. In some embodiments, the B-cells are not cancerouscells.

The present invention further provides pharmaceutical compositionscomprising one or more of the CD37-binding agents described herein. Incertain embodiments, the pharmaceutical compositions further comprise apharmaceutically acceptable vehicle. These pharmaceutical compositionsfind use in treating autoimmune and inflammatory disease in humanpatients.

In certain embodiments, formulations are prepared for storage and use bycombining a purified antibody or agent of the present invention with apharmaceutically acceptable vehicle (e.g. carrier, excipient)(Remington, The Science and Practice of Pharmacy 20th Edition MackPublishing, 2000). Suitable pharmaceutically acceptable vehiclesinclude, but are not limited to, nontoxic buffers such as phosphate,citrate, and other organic acids; salts such as sodium chloride;antioxidants including ascorbic acid and methionine; preservatives (e.g.octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens, such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight polypeptides (e.g. less than about 10 amino acid residues);proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilicpolymers such as polyvinylpyrrolidone; amino acids such as glycine,glutamine, asparagine, histidine, arginine, or lysine; carbohydratessuch as monosaccharides, disaccharides, glucose, mannose, or dextrins;chelating agents such as EDTA; sugars such as sucrose, mannitol,trehalose or sorbitol; salt-forming counter-ions such as sodium; metalcomplexes (e.g. Zn-protein complexes); and non-ionic surfactants such asTWEEN or polyethylene glycol (PEG).

The pharmaceutical compositions of the present invention can beadministered in any number of ways for either local or systemictreatment. Administration can be topical (such as to mucous membranesincluding vaginal and rectal delivery) such as transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders; pulmonary (e.g., by inhalation or insufflation of powdersor aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal); oral; or parenteral including intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial (e.g., intrathecal or intraventricular)administration.

An antibody or immunoconjugate of the invention can be combined in apharmaceutical combination formulation, or dosing regimen as combinationtherapy, with a second compound having anti-autoimmune or inflammatoryproperties. The second compound of the pharmaceutical combinationformulation or dosing regimen can have complementary activities toCD37-binding agent of the combination such that they do not adverselyaffect each other. Pharmaceutical compositions comprising theCD37-binding agent and the second agent are also provided. For example,CD37-binding agents can be administered in combination with CD20-bindingagents, such as Rituximab. In other embodiments, CD37-binding agents canbe administered in combination with salicylate; nonsteroidalanti-inflammatory drugs such as indomethacin, phenylbutazone,phenylacetic acid derivatives (e.g., ibuprofen and fenoprofen),naphthalene acetic acids (naproxen), pyrrolealkanoic acid (tometin),indoleacetic acids (sulindac), halogenated anthranilic acid(meclofenamate sodium), piroxicam, zomepirac and diflunisal;antimalarials such as chloroquine; gold salts; penicillamine; orimmunosuppressive agents such as methotrexate or corticosteroids. Insome embodiments, the CD37-binding agent is administered in combinationwith a second therapeutic selected from the group consisting ofmethotrexate, an anti-CD20 therapeutic, an anti-IL-6 receptortherapeutic, an anti-IL-12/23p40 therapeutic, a chemotherapeutic, animmunosuppressant, an anti-interferon beta-1a therapeutic, glatirameracetate, an anti-α4-integrin therapeutic, fingolimod, an anti-BLystherapeutic, CTLA-Fc, or an anti-TNF therapeutic. In some embodiments,the CD37-binding agent is administered in combination with a secondtherapeutic that is an antibody directed against an antigen selectedfrom a group consisting of CD3, CD14, CD19, CD20, CD22, CD25, CD28,CD30, CD33, CD36, CD38, CD40, CD44, CD52, CD55, CD59, CD56, CD70, CD79,CD80, CD103, CD134, CD137, CD138, and CD152. In some embodiments, theCD37-binding agent is administered in combination with a secondtherapeutic that is an antibody directed against a target selected fromthe group consisting of IL-2, IL-6, IL-12, IL-23, IL-12/23 p40, IL-17,IFNγ, TNFα, IFNα, IL-15, IL-21, IL-1a, IL-1b, IL-18, IL-8, IL-4, GM-CSF,IL-3, and IL-5. In some embodiments, the CD37-binding agents areadministered in combination with methotrexate.

For the treatment of the disease, the appropriate dosage of an antibodyor agent of the present invention depends on the type of disease to betreated, the severity and course of the disease, the responsiveness ofthe disease, whether the antibody or agent is administered fortherapeutic or preventative purposes, previous therapy, patient'sclinical history, and so on all at the discretion of the treatingphysician. The antibody or agent can be administered one time or over aseries of treatments lasting from several days to several months, oruntil a cure is affected or a diminution of the disease state isachieved. Optimal dosing schedules can be calculated from measurementsof drug accumulation in the body of the patient and will vary dependingon the relative potency of an individual antibody or agent. Theadministering physician can easily determine optimum dosages, dosingmethodologies and repetition rates. In certain embodiments, dosage isfrom 0.01 μg to 100 mg per kg of body weight, and can be given once ormore daily, weekly, monthly or yearly. In certain embodiments, theantibody or other CD37-binding agent is given once every two weeks oronce every three weeks. In certain embodiments, the dosage of theantibody or other CD37-binding agent is from about 0.1 mg to about 20 mgper kg of body weight. The treating physician can estimate repetitionrates for dosing based on measured residence times and concentrations ofthe drug in bodily fluids or tissues.

The combination therapy can provide “synergy” and prove “synergistic”,i.e. the effect achieved when the active ingredients used together isgreater than the sum of the effects that results from using thecompounds separately. A synergistic effect can be attained when theactive ingredients are: (1) co-formulated and administered or deliveredsimultaneously in a combined, unit dosage formulation; (2) delivered byalternation or in parallel as separate formulations; or (3) by someother regimen. When delivered in alternation therapy, a synergisticeffect can be attained when the compounds are administered or deliveredsequentially, e.g. by different injections in separate syringes. Ingeneral, during alternation therapy, an effective dosage of each activeingredient is administered sequentially, i.e. serially, whereas incombination therapy, effective dosages of two or more active ingredientsare administered together.

VI. Kits Comprising CD37-Binding Agents

The present invention provides kits that comprise the antibodies,immunoconjugates or other agents described herein and that can be usedto perform the methods described herein. In certain embodiments, a kitcomprises at least one purified antibody against CD37 in one or morecontainers. In some embodiments, the kits contain all of the componentsnecessary and/or sufficient to perform a detection assay, including allcontrols, directions for performing assays, and any necessary softwarefor analysis and presentation of results. A label or indicatordescribing, or a set of instructions for use of, kit components in aligand detection method of the present invention, can also be included.The instructions may be associated with a package insert and/or thepackaging of the kit or the components thereof. One skilled in the artwill readily recognize that the disclosed antibodies, immunoconjugatesor other agents of the present invention can be readily incorporatedinto one of the established kit formats which are well known in the art.Such kits can also include, for example, other compounds and/orcompositions, a device(s) for administering the compounds and/orcompositions, and written instructions in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products.

Further provided are kits comprising a CD37-binding agent (e.g., aCD37-binding antibody), as well as a second agent. In certainembodiments, the second agent is rituximab. In certain embodiments, thesecond agent is methotrexate.

Embodiments of the present disclosure can be further defined byreference to the following non-limiting examples, which describe indetail preparation of certain antibodies of the present disclosure andmethods for using antibodies of the present disclosure. It will beapparent to those skilled in the art that many modifications, both tomaterials and methods, can be practiced without departing from the scopeof the present disclosure.

EXAMPLES

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application.

All publications, patents, patent applications, internet sites, andaccession numbers/database sequences (including both polynucleotide andpolypeptide sequences) cited herein are hereby incorporated by referencein their entirety for all purposes to the same extent as if eachindividual publication, patent, patent application, internet site, oraccession number/database sequence were specifically and individuallyindicated to be so incorporated by reference.

Example 1 CD37 Expression in Normal Human PBMCs

The CD37 antigen was reported to be expressed on B-cells from the pre-Bstage to the peripheral mature B-cell stage, while being absent onB-cell progenitors and terminally differentiated plasma cells. (Link etal., 1987, J Pathol. 152:12-21). In addition, the CD37 antigen is onlyweakly expressed on T-cells, myeloid cells and granulocytes(Schwartz-Albiez et al. 1988, J. Immunol., 140(3)905-914).

The ability of antibodies (including certain CD37 antibodies andimmunoconjugates previously described in U.S. Published Application No.2011/0256153, which is herein incorporated by reference in its entirety)to bind to normal human B-cells was measured using flow cytometry assayswith fluorescently labeled antibodies. In addition, the commerciallyavailable QuantiBRITE system from BD Biosciences was used to estimateantigen density based on the number of antibodies bound to the cells(ABC). The QuantiBRITE system from BD Biosciences utilizes the followingreagents: anti-CD20-PE supplied at 100 μg/mL and QuantiBRITE PE suppliedas lyophilized PE-labeled beads. In addition, the huCD37-3 antibody waslabeled with PE to obtain an antibody-PE conjugate with an Ab:PE ratioof approximately 1:1.

Fresh buffy coats from healthy donors were obtained from Research BloodComponents (Brighton, Mass., US) as a source of normal blood cells.Buffy coats were prepared by centrifugation of a unit of whole blood andcollecting the interface between the plasma and the red blood cells.This unpurified buffy coat contains PBMCs, neutrophils, platelets, redblood cells, and plasma and was used for experiments on the same day itwas drawn. Peripheral blood mononuclear cells (PBMCs) were prepared frombuffy coats by standard density gradient centrifugation usingFicoll-Paque as follows. Blood was diluted 1:3 with 1×HBSS containing 5mM EDTA and up to 30 mL were added to a 50 mL conical tube. Ten mL ofFicoll-Paque (GE Healthcare) were slowly added to the bottom of eachtube. Samples were centrifuged at 500×g with no brake at RT for 30minutes to obtain a layer of PBMCs below the plasma and to remove redblood cells and most granulocytes. The PBMCs were transferred to newtubes and washed twice with 1×HBSS containing 5 mM EDTA bycentrifugation at 400×g for 10 minutes at RT. Staining buffer (1×HBSS,1% BSA, 0.1% sodium azide) was then used to resuspend the PBMC pelletsat 6.25×10⁶ cells/mL. Eighty μL of cells were transferred to around-bottom 96-well plate to achieve 5×10⁵ cells/assay and 20 μL ofhuman serum (Sigma H4522) were added to block Fc receptor-mediatedbinding and incubated with cells on ice for 20 min in the dark.Fluorescently labeled antibodies obtained from Miltenyi were used toidentify PBMC populations: anti-CD3-allophycocyanin (APC) was used toidentify T-cells, anti-CD19-APC for B-cells, anti-CD56-APC for naturalkiller (NK) cells and anti-CD14-APC for monocytes.

Cells were co-stained for CD37 expression using 20 μL of huCD37-3-PE fora final concentration of approximately 10 μg/mL. Likewise, cells wereco-stained for CD20 expression using 20 μL of anti-CD20-PE. As a controla non-binding PE-labeled huIgG1 isotype control antibody was used at 10μg/mL. Staining was carried out for 1 hour on ice in the dark. Sampleswere washed twice with staining buffer and fixed in 200 μL of 1%formaldehyde in 1×PBS. Samples were stored at 4° C. in the dark untilacquisition, which was performed within 4 days of sample preparation.

A fresh tube of QuantiBRITE beads was reconstituted in the supplied tubewith 0.5 mL of staining buffer just prior to sample acquisition. Sampleswere acquired on a FACSCalibur flow cytometer (BD Biosciences).Compensation controls were run with each assay to select appropriateinstrument settings and at least 10,000 events were collected for eachsample. Instrument settings for fluorescence and compensation were keptthe same for both cell sample and bead sample acquisition to allow foran accurate comparison. CellQuest (version 5.2.1, BD Biosciences) wasused for acquisition control and analysis.

The QuantiBRITE analysis utilizes on a bead standard with 4 beadpopulations conjugated with a known number of PE molecules. For dataanalysis, a G1 gate was drawn around the bead singlets on an FSC-H/SSC-Hscatter plot. This gated bead population was subsequently analyzed usinga histogram plot of FL2-H to evaluate the level of PE staining. Separatemarkers were drawn around the peaks of the four bead populations (M1-M4)and the geometric mean for FL2 of each bead population was determined.The FL2 geometric mean of each bead was plotted against the lot specificPE/bead values in a log-log plot. Linear regression was performed toobtain a standard curve using the following equation: y=mx+c, with “m”equal to the slope and “c” equal to the y-intercept.

For PBMC sample analysis, a G1 gate was drawn around the positivefluorescent cell population of interest on an SSC-H/FL4-H dot plot. Thisgated cell population was subsequently analyzed using a histogram plotof FL2-H to evaluate the level of PE-labeled antibody staining. The FL2geometric mean was determined for each blood cell sample stained withanti-CD37-PE or anti-CD20-PE, as well as unstained control samples. Allgeometric mean values for FL2 were plotted against the bead standardcurve and values for PE per cell were extrapolated. Since bothantibody-PE conjugates were at a PE:Ab ratio of approximately 1:1, thevalues for PE per cell correspond to the number of antibodies bound percell (ABC) value. Experiments were performed with duplicate samples foreach assay. The mean and standard deviation was determined from severalassays for each blood cell population.

CD37 expression was evaluated in normal blood cells from 4 independentdonors. Results were compared to CD20 staining, unstained cells and anon-binding huIgG-PE conjugate as controls. An example of a typicalstaining profile of normal B-cells is given in a histograms in FIG. 1.The average ABC values of 4 different experiments for CD37 and CD20 werecalculated and listed in Table 1.

TABLE 1 ABC values for CD37 and CD20 expression on human PBMC samples NoAb huIgG-PE CD37 ABC CD20 ABC control control CD19+ B-cells 77,44094,598 80 76 CD3+ T cells 2,016 336 74 68 CD56+ NK cells 3,090 264 85 88CD14+ monocytes 5,244 794 180 215

The highest overall CD37 staining level was found in CD19+ B-cells atapproximately 77,000 ABC. In addition, CD37 staining was seen at lowlevels in other PBMC populations examined, with CD14+ monocytes showingCD37 staining at approximately 5,000 ABC, CD56+ NK cells at 3,000 ABC,and CD3+ T cells at 2,000 ABC. Staining with the non-binding huIgG-PEcontrol resulted in ABC values of approximately 70-90 for B, T and NKcells and approximately 200 for monocytes. In the same 4 donors CD20expression was evaluated in comparison to CD37. In accordance withpublished findings, the CD20 staining was restricted mainly to CD19+B-cells with an ABC value of approximately 95,000 ABC. The CD20expression level was just slightly higher than the CD37 expressionlevel. Only minimal CD20 staining was observed in other PBMC populationsexamined, with CD14+ monocytes showing CD20 staining at 794 ABC, CD56+NK cells at 264 ABC and CD3+ T cells at 336 ABC.

This result demonstrates that high CD37 expression is mainly restrictedto B-cells in peripheral blood samples with only minor expression onperipheral T cells, NK cells and monocytes. This is consistent withpublished findings ((Moore et al. 1986, J Immunol. 137(9):3013-8;Schwartz-Albiez et al. 1988, J. Immunol., 140(3)905-914). In addition,we found that the CD37 expression levels on peripheral B-cells issimilar to the level of CD20 expression. This expression patternstrongly suggest that CD37 directed therapies may be a suitable fortargeting B-cells in diseases such as B-cell malignancies, autoimmunediseases, inflammatory diseases or other disorders of the immune systemanalogous to the use of CD20 directed therapies.

Example 2A In Vitro B-Cell Depletion Using Purified PBMCs

The ability of humanized antibodies to deplete B-cells was measuredusing in vitro assays with human PBMCs according to published studiesperformed with rituximab (Vugmeyster et al. Cytometry A. 2003;52(2):101-9 and Vugmeyster et al. Int Immunopharmacol. 2004;4(8):1117-24). Alemtuzumab (Campath) was used as appositive control,since it has been reported to efficiently deplete lymphocytes in vivoand in vitro (Hale, Blood. 1983 October; 62(4):873-82 and Waldmann,Philos Trans R Soc Lond B Biol Sci. 2005 Sep. 29; 360(1461):1707-11).

Fresh buffy coats from healthy donors were obtained from Research BloodComponents (Brighton, Mass., US) as a source of normal blood cells forall experiments within this study. Buffy coats were prepared bycentrifugation of a unit of whole blood and collecting the interfacebetween the plasma and the red blood cells. This unpurified buffy coatcontains PBMCs, neutrophils, platelets, red blood cells, and plasma andwas used for experiments on the same day it was drawn. Peripheral bloodmononuclear cells (PBMCs) were prepared from buffy coats by standarddensity gradient centrifugation using Ficoll-Paque as follows. Blood wasdiluted 1:3 with 1×HBSS containing 5 mM EDTA and up to 30 mL were addedto a 50 mL conical tube. Ten mL of Ficoll-Paque (GE Healthcare) wereslowly added to the bottom of each tube. Samples were centrifuged at500×g with no brake at RT for 30 minutes to obtain a layer of PBMCsbelow the plasma and to remove red blood cells and most granulocytes.The PBMCs were transferred to new tubes and washed twice with 1×HBSScontaining 5 mM EDTA by centrifugation at 400×g for 10 minutes at RT.Staining buffer (1×HBSS, 1% BSA, 0.1% sodium azide) was then used toresuspend the PBMC pellets in the initial blood volume to achieve theoriginal cell density.

To assess the effect of huCD37-3, huCD37-3-SMCC-DM1, huCD37-50,huCD37-50-SMCC-DM1, rituximab, alemtuzumab (Campath), and TRU-016 onPBMC depletion, 90 μL of purified cells were added to 12×75 mmpolystyrene tubes and incubated with 10 μL of a 100 μg/mL solution ofeach sample or a huIgG isotype control antibody for 1 hr at 37° C. in ahumidified 5% CO₂ incubator. The final antibody (Ab) concentration was10 μg/mL in a final volume of 100 μL in staining buffer. Threeindependent samples were prepared for each treatment.

To identify populations of PBMCs, all samples were co-stainedimmediately after Ab incubation with 10-20 μL of fluorescently labeledAbs obtained from, for example, BD Biosciences or Miltenyi.Anti-CD3-PerCP-Cy5.5 was used to identify T cells, anti-CD19-APC forB-cells, and anti-CD14-FITC for monocytes. Staining was carried out in atotal of 150 μL for 30 min in the dark at RT. CountBright AbsoluteCounting Beads (Invitrogen) were vortexed and added to each sample at 50μL per tube. For PBMC prep samples, cells were washed once with 1 mLstaining buffer and centrifuged at 400×g for 3-5 min. Supernatant wasremoved with a 1 mL pipette and cells were resuspended in 500 μL of 1%formaldehyde in 1×PBS. Samples were stored at 4° C. in the dark untilacquisition, which was performed within 4 days of sample preparation.

TreeStar FlowJo software (version PC 7.5) was used for data analysis. Agate was drawn around the CountBright bead population on an FSC-H vsSSC-H dot plot to determine a total bead count for the sample. Todetermine the total count for each PBMC population of interest, aseparate gate was drawn around the positive fluorescent population on anSSC-H vs FL(x)-H dot plot, where x is the channel of interest.Specifically, a total count for T cells in a sample was found by gatingthe positive population on an SSC-H vs FL3-H dot plot; for B-cells, thepositive population was found on an SSC-H vs FL4-H dot plot; for NKcells, an SSC-H vs FL2-H dot plot was used; for monocytes, an SSC-H vsFL1-H dot plot was used. The ratio of CD19+ cells for B-cells (CD3+cells for T cells, CD56+ cells for NK cells, or CD14+ cells formonocytes) relative to beads was determined and multiplied by 100.Percent depletion was then calculated by taking the ratio of the cell tobead ratio in treated samples relative to the cell to bead ratio inisotype control treated samples, subtracting this from 1 and multiplyingby 100. This corresponds to the following formula: PercentDepletion=100×(1−cell to bead ratio of treated sample/cell to bead ratioof control sample). Data for all cell types was analyzed in the samemanner.

For two donors tested, treatment of purified PBMC samples with huCD37-3,huCD37-3-SMCC-DM1, huCD37-50 or huCD37-50-SMCC-DM1 resulted inapproximately 55-70% depletion of B-cells (see FIG. 2). There was lessthan 10% depletion of T cells or monocytes. The B-cell restricteddepletion effect indicates that this activity is linked to the high CD37expression on B-cells. In comparison, treatment with the anti-CD20antibody rituximab resulted in approximately 30-40% depletion ofB-cells. Treatment with the anti-CD37 SMIP™ TRU-016 resulted in only20-30% depletion of B-cells. Alemtuzumab treatment resulted in depletionof 60-70% of B-cells, 55-65% of T cells and 40-65% of monocytes.

Example 2B Dose Response for In Vitro B-Cell Depletion Using PurifiedPBMCs

To evaluate the dose-response of the antibodies and conjugates, purifiedPBMCs from 2 donors were incubated with a 5-fold sample dilution series.Each sample dilution was added at 10 μL per tube to 90 μL of purifiedcells in triplicate and incubated for 1 hour at 37° C. in a humidified5% CO₂ incubator. The final concentration ranged from 10 μg/mL to 0.13ng/mL. The same amount of a non-binding huIgG Ab was used as an isotypecontrol.

For two donors tested, treatment of purified PBMC samples withhuCD37-3-SMCC-DM1 resulted in a clear dose-response for the B-celldepletion activity (see FIGS. 3A and B). Incubation withhuCD37-3-SMCC-DM1 caused in vitro depletion of approximately 60% ofB-cells with an EC50 of 40-75 ng/mL. For an additional donor tested,treatment of purified PBMC samples with huCD37-3, huCD37-38, huCD37-50,and huCD37-56 antibodies also resulted in a clear dose-response for theB-cell depletion activity (see FIG. 3C). Incubation with theseantibodies caused in vitro depletion of approximately 60-70% of B-cellswith an EC50 of 20-30 ng/mL.

Example 2C In Vitro B-Cell Depletion Using Whole Blood

The ability of humanized antibodies to deplete B-cells was measuredusing in vitro assays with whole blood according to published studiesperformed with rituximab (Vugmeyster et al. Cytometry A. 2003;52(2):101-9 and Vugmeyster et al. Int Immunopharmacol. 2004;4(8):1117-24).

Fresh buffy coats from healthy donors were obtained from Research BloodComponents (Brighton, Mass., US) as a source of normal blood cells forall experiments within this study. To assess the effect of huCD37-3,huCD37-3-SMCC-DM1, rituximab, alemtuzumab (Campath), and TRU-016 onperipheral blood cells (PBCs) in a whole blood matrix, 90 μL of wholeblood from a buffy coat were incubated with Abs or isotype control asdetailed above in a total volume of 100 μL. Three independent sampleswere prepared for each Ab treatment.

To identify populations of blood cells, all samples were co-stainedimmediately after Ab incubation with 10-20 μL of fluorescently labeledAbs obtained from, for example, BD Biosciences or Miltenyi.Anti-CD3-PerCP-Cy5.5 was used to identify T cells, anti-CD19-APC forB-cells, anti-CD56-PE for NK cells, and anti-CD14-FITC for monocytes.Staining was carried out in a total of 150 μL for 30 min in the dark atRT. CountBright Absolute Counting Beads (Invitrogen #C36950) werevortexed and added to each sample at 50 μL per tube to allowstandardization of cell counts.

Following cell staining, 2 mL of BD FACS Lysing Solution (BDBiosciences, diluted 1:10 in dH₂O according to the manufacturer'sinstructions) were added to each sample in order to lyse the RBCspresent. Samples were incubated at RT for 15-20 min in the dark,centrifuged at 400×g for 3-5 min, and resuspended in 500 μL of 1%formaldehyde in 1×PBS. Samples were stored at 4° C. in the dark untilacquisition, which was performed within 4 days of sample preparation.Samples were acquired on a BD FACSCalibur. Compensation controls wererun with each assay to confirm instrument settings. A total of 160,000ungated events were acquired for each sample using BD CellQuest software(version 5.2). TreeStar FlowJo software (version PC 7.5) was used fordata analysis as described above.

For one donors tested, treatment of purified PBMC samples with huCD37-3,huCD37-3-SMCC-DM1, huCD37-50 or huCD37-50-SMCC-DM1 resulted inapproximately 40% depletion of B-cells (see FIG. 4). There was less than10% depletion of T cells, NK cells or monocytes. As seen for purifiedPBMCs, the in vitro depletion is restricted to B-cells indicating thatthe activity is linked to the high CD37 expression on B-cells. Incomparison, treatment with the anti-CD20 antibody rituximab or theanti-CD37 SMIP™ TRU-016 resulted in a less than 10% depletion ofB-cells. Alemtuzumab treatment resulted in depletion of 40% of B-cells,80% of T cells, 15% of NK cells and 20% of monocytes.

Example 2D Dose Response for In Vitro B-Cell Depletion Using Whole Blood

To evaluate the dose-response of the antibodies and conjugates, wholeblood from 2 donors was incubated with a 10-fold sample dilution series.Each sample dilution was added at 10 μL per tube to 90 μL of purifiedcells in triplicate and incubated for 1 hr at 37° C. in a humidified 5%CO₂ incubator. The final concentration ranged from 10 μg/mL to 0.1ng/mL. The same amount of a non-binding huIgG Ab was used as an isotypecontrol.

For two donors tested, treatment of whole blood samples with huCD37-3 orhuCD37-3-SMCC-DM1 resulted in a clear dose response for the B-celldepletion activity (see FIGS. 5A and B). In addition, huCD37-50 wastested for one donor and also showed a similar dose response for theB-cell depletion activity (see FIG. 5B). Incubation with huCD37-3,huCD37-3-SMCC-DM1 or huCD37-50 caused a maximum response of in vitrodepletion of approximately 30-45% of B-cells with an EC50 of 40-120ng/mL.

In addition to the in vitro experiment described above, the capacity ofCD37 antibodies to deplete B cells in vivo can be tested in huCD37expressing mice (described in Example 3) and, for antibodies thatcrossreact with macaque CD37, in monkey.

Example 2E In Vitro Cytokine Release Studies Using Human PBMCs

In vitro cytokine release was measured by ELISpot for IFN-γ(Interferon), TNF-α (Tumor Necrosis Factor) and IL-6 (Interleukin-6)using peripheral blood mononuclear cells (PBMCs) from healthy humandonors incubated for 18-20 hours with compounds at a concentration of2.5 ng/mL to 250 μg/mL. The ELISpot method is designed to measure thenumber of cells secreting cytokine by capturing the cytokine onto theassay plate during the entire length of the incubation. In all assaysthe positive control anti-CD3 antibody CD3-2, as well as a negativenon-binding isotype huIgG control antibody was included. Alemtuzumab(Campath®) and rituximab (Rituxan®) were used in comparison, since bothhave been reported to induce cytokine release in patients (Wing. J ClinInvest. 98:2819-26 (1996) and Winkler, Blood 94:2217-2224 (1999)). Theassay conditions were chosen to reflect conditions that are relevant forantibody therapeutics. The highest concentration of 250 μg/mL testedcorresponds to the maximum serum concentration of an antibody, such asfor example the CD20-directed rituximab, in patient plasma after aninfusion of 10 mg/kg of antibody.

As can be seen in FIGS. 6 and 7, the positive control anti-CD3 antibodyinduced release of very high levels of IFN-γ, TNF-α and IL-6 with PBMCsfrom two different donors. In the same assays, alemtuzumab causedintermediate cytokine release, while rituximab caused moderate cytokinerelease with PBMCs from two different donors. In contrast, huCD37-3,huCD37-50, huCD37-3-SMCC-DM1 or huCD37-50-SMCC-DM1 did not causesignificant cytokine release in our assays.

This underscores the utility of the described CD37-targeting antibodiesor conjugates as therapeutics as they combine potent activity, such asB-cell depletion, with a favorable safety profile with respect tocytokine release.

Example 3 In Vivo Models to Evaluate the Activity of CD37 DirectedAntibodies or Conjugates

B-cell depletion is known to ameliorate autoimmune diseases. In fact,rituximab has been approved for rheumatoid arthritis treatment (EdwardsJ C et al. Nat Rev Immunol. 6: 119 (2006)). In animal models, B-celldepletion using antibodies against B-cell antigens such as CD20, CD19and CD79 has been shown to inhibit or ameliorate several autoimmunediseases including systemic lupus erythematosus (SLE), experimentalautoimmune encephalomyelitis (EAE; mouse model of multiple sclerosis),type-1 diabetes (T1D) and rheumatoid arthritis (RA). The CD37 antigen isexpressed at high levels in human B-cells. Therefore, antibodies orimmunoconjugates directed against the CD37 antigen could potentiallydeplete B-cells and be therefore useful to treat multiple autoimmunediseases.

To test the utility of CD37 targeting antibodies and immunoconjugates totreat human autoimmune diseases, the activity of such CD37 targetingantibodies and immunoconjugates can be studied in mice using severalmurine autoimmune disease models.

For example, anti-murine CD37 antibodies can be generated usingCD37-knock-out mice or other species such as rat and hamster, andantibodies that deplete B-cell in vivo effectively can be selected. Thetherapeutic potential of anti-CD37 antibodies can be tested in mousemodels representing human autoimmune diseases, for example, aspontaneous T1D model in NOD mice, a myelin oligodendrocyte glycoprotein(MOG) peptide induced EAE model in wild type C57/B16 mice, a collageninduced rheumatoid arthritis model in DBA/1 mice or a spontaneoussystemic lupus erythematosus (SLE) model in MRL/lpr mice. Examples ofmurine CD37 antibodies and their therapeutic efficacy in various animalmodels of autoimmune disease are provided below.

Alternatively, the therapeutic potential of anti-human CD37 antibodiesand immunoconjugates can also be tested in murine autoimmune diseasemodels that have been engineered to express the human CD37 antigen. Suchhuman CD37 (huCD37) expressing mice can be generated using standardknock in (KI) or transgenic (Tg) approaches. For example, to generatehuCD37 KI mice, human CD37 cDNA can be inserted into the murine CD37locus in the C57/B16 embryonic stem (ES) cells. The homozygous huCD37 KImice will express human CD37 cDNA under the regulation of the endogenousmurine CD37 promoter, thus the expression pattern of the huCD37 wouldmimic that of the endogenous muCD37. The different approach utilizesbacterial artificial chromosome (BAC) containing the human CD37 genethat can be randomly inserted into the mouse genome. This transgenicapproach has been used successfully to generate huCD20 Tg mice resultingin B-cell specific high level expression of the antigen.

The resulting huCD37 expressing mice based on the C57/B16 background canbe used to further develop several autoimmune disease model. Forexamples, MOG peptide immunization in the C57/B16 strain background caninduces severe EAE in two weeks. In addition, introducing a FcγRIIBknock out phenotype by breeding huCD37 expressing mice with C57/B16FcγRIIB knock out mice should yield a mouse model that spontaneouslydevelop SLE and develop RA upon immunization with collagen II antigen.Alternatively, backcrossing of the huCD37 expressing C57/B16 mice intothe NOD or MRL/lpr background for 10 generations can provide spontaneousT1D and SLE models, respectively.

Example 4A Generation of Anti-muCD37 Monoclonal Antibody Clone 252-3

To develop proof of concept that CD37 targeting antibody andimmunoconjugate can inhibit autoimmune disease, anti-murine CD37(muCD37) monoclonal antibodies were generated by immunizingCD37-knock-out C57Bl/6 mice with 300-19, a murine pre-B cell line thatendogenously expresses the muCD37 antigen. The immunogen was injectedsubcutaneously at the dose of 5×10⁶ cells per mouse every 2 weeks for 5times. Three days before being sacrificed for hybridoma generation, theimmunized mice received intraperitoneal injection of another dose ofantigen. The spleen cells were fused with murine myeloma P3X63Ag8.653cells (P3 cells) (J. F. Kearney et al. 1979, J Immunol, 123: 1548-1550)at ratio of 1 P3 cells:3 spleen cells according to standard procedure.The fused cells were cultured in RPMI-1640 selection medium containinghypoxanthine-aminopterin-thymidine (HAT) (Sigma Aldrich) in 5% CO₂incubator at 37° C. until hybridoma clones were ready for antibodyscreening.

Screening was done using flow cytometric binding assay with spleen cellsfrom wild type mice and CD37-knock-out mice. The spleen cells werecounterstained with anti-CD45R (B220) antibody to identify B cells thatconstitutively express CD37 antigen. The hybridomas producing antibodythat bound the wild type, but not CD37-knock-out, B cells were subclonedby limiting dilution. One stable subclone (clone 252-3) was obtained.The 252-3 hybridoma was expanded in low IgG serum containing media andthe antibody was purified using standard methods with protein A/Gchromatography.

Example 4B Characterization of Anti-muCD37 Monoclonal Antibody Clone252-3

The purified 252-3 monoclonal antibody was identified as a mouse IgG2awith IsoStrip mouse monoclonal antibody isotyping kit (Roche DiagnosticsCorporation, Indianapolis, Ind.). To determine the binding affinity tothe muCD37 antigen, various concentrations of 252-3 antibody wereincubated with 300-19 cells, a murine pre-B cell line that expresses themuCD37 antigen, for 30 minutes at 4° C. Cells were then washed andcounterstained with anti muIgG-PE conjugate (Jackson Immunoresearch,West Grove, Pa.) for 30 minutes at 4° C. The cells were finally washed,fixed in formalin and analyzed by flow cytometry using a FACSarray (BDBioscience, San Jose, Calif.). The flow cytometry data were analyzedusing FlowJo (Tree Star Inc., Ashland, Oreg.) and the geometric meanfluorescence intensity was plotted against the antibody concentration ina semi-log plot (FIG. 8). A dose-response curve was generated bynon-linear regression and the EC50 value of the curve, which correspondsto the apparent dissociation constant (Kd) of the antibody, wascalculated using GraphPad Prism (GraphPad Software Inc., La Jolla,Calif.). It was found that the Kd of the 252-3 antibody was 14 nM. Incontrast, the 252-3 antibody did not bind to human tumor cellsexpressing the human CD37 antigen. The 252-3 antibody was then used as asurrogate antibody in murine autoimmune disease models to demonstratethe therapeutic potential of a CD37-targeting antibody for the treatmentof autoimmune diseases (Examples 5-7).

Example 5 Anti-muCD37 Monoclonal Antibody Inhibits ExperimentalAutoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an animal model ofinflammatory demyelinating disease of the central nervous system (CNS),including multiple sclerosis in human. Murine EAE is commonly induced byimmunization of spinal cord homogenates, brain extracts, or CNS proteinsuch as myelin protein or peptide, followed by injection of pertussistoxin to break down the blood-brain barrier and allow immune cellsaccess to the CNS tissue. This immunization leads to multiple smalldisseminated lesions of demyelination in the brain and spinal cord,causing tail paralysis followed by limb paralysis.

To test the activity of anti-muCD37 antibody in the EAE model, we firststudied the capacity of the 252-3 antibody to deplete B cells in vivo.C57Bl/6 mice were injected intraperitoneally with 25 mg/kg of 252-3antibody or polyclonal murine IgG (Jackson Immunoresearch, West Grove,Pa.) as a control. Peripheral blood was collected at different timepoints and analyzed for B and T cell levels by flow cytometry.Allophycocyanin (APC)-conjugated anti-mouse CD45R (B220) antibody(ebioscience, San Diego, Calif.) and fluorescein isothiocyanate(FITC)-conjugated anti CD3c antibody (ebioscience, San Diego, Calif.)were used to stain B and T cell populations, respectively. B celldepletion was assessed by calculating the ratio of B to T cells for eachsample and the B/T ratio was normalized by setting the average B/T ratioof murine IgG-treated samples to 100%. The normalized B/T cell ratio wasplotted for muIgG control mice and 252-3 antibody treated mice (FIG.9A). The result show that the B cell level of the mice treated with252-3 antibody was rapidly reduced within a few hours after the antibodyinjection. The B cell depletion reached ˜70% at 3 h and peaked at day 3(>95%). After day 3, the B cell level slowly increased and reached ˜60%of the normal level at day 14. This data suggests that the 252-3antibody can rapidly and efficiently deplete peripheral blood B cells,and this effect was sustained for at least 7 days after the antibodyinjection.

The second study tested the capacity of 252-3 antibody to inhibit EAE.In this study, EAE was induced in C57Bl/6 mice by subcutaneousimmunization of MOG₃₅₋₅₅ peptide emulsified in complete Freund'sadjuvant (EAE kit from Hooke Laboratories, Lawrence, Mass.) into theupper and lower back at day 0 and two intraperitoneal injections ofpertussis toxin at 2 h and 24 h after antigen immunization. Mice werechecked for EAE signs daily starting on day 7 after immunization. Thedisease severity was scored on a scale of 0 to 5 using the followingcriteria:

Score Clinical Observations 0 No obvious changes in motor functions ofthe mouse in comparison to non-immunized mice. When picked up by thetail, the tail has tension and is erect. Hind legs are usually spreadapart. When the mouse is walking, there is no gait or head tilting. 1Limp tail. When the mouse is picked up by tail, instead of being erect,the whole tail drapes over your finger. 2 Limp tail and weakness of hindlegs. When the mouse is picked up by tail, legs are not spread apart,but held closer together. When the mouse is observed when walking, ithas clearly apparent wobbly walk. 3 Limp tail and complete paralysis ofhind legs (most common) OR, Limb tail with paralysis of one front andone hind leg. OR, ALL of: Severe head tilting Walking only along theedges of the cage Pushing against the cage wall Spinning when picked upby the tail 4 Limp tail, complete hind leg and partial front legparalysis. Mouse is minimally moving around the cage but appears alertand feeding. Usually, euthanasia is recommended after the mouse scoreslevel 4 for 2 days. When the mouse is euthanized because of severeparalysis, a score of 5 is entered for that mouse for the rest of theexperiment. 5 Complete hind and front leg paralysis, no movement aroundthe cage. OR, Mouse is spontaneously rolling in the cage. OR, Mouse isfound dead due to paralysis. If mouse is alive, euthanize the mouseimmediately if it scores 5. Once mouse scored 5, the same score isentered for all the days for the rest of the experiment.

All mice started to show signs of EAE between 12 to 18 days afterantigen immunization. At the disease onset, mice were randomized and the252-3 antibody or polyclonal muIgG was injected once intraperitoneallyat a 25 mg/kg dose. A total of 10 mice were enrolled for each group. Atthe end of the study (18 days after the disease onset), the data weresynchronized based on the day of disease onset for each mouse. Thedisease progression plot (FIG. 9B) shows that mice from both groups hadrelapsing-remitting form of EAE. During the first wave of clinicalsymptoms, the control mice reached the mean of 3 while the mice treatedwith 252-3 antibody had a mean of 2. The difference in disease severitybetween these two groups was sustained for more than 2 weeks after thedisease onset. Taken together, this data suggests that the 252-2antibody treatment rapidly depletes the B cell population and alleviatesEAE.

Example 6 Anti-muCD37 Monoclonal Antibody Inhibits Type-1 Diabetes inNOD Mice

Type-1 diabetes (T1D) or juvenile diabetes or insulin-dependent diabetesmillitus (IDDM) is caused by auto-immune reaction againstinsulin-producing pancreatic beta cells. Destruction of beta cellsreduces insulin production and increases glucose level that producesvarious clinical symptoms. T1D incidence in Northern Europe and the USis between 8 and 17/100,000. Insulin supplement is the most commontreatment of the disease.

Non-obese diabetic (NOD) mice spontaneously develop T1D and have beenwidely used to model the human disease. In NOD mice, the disease startswith leukocytic infiltration of the pancreatic islets (called insulitis)as early as 4 weeks of age. The insulitis progresses rapidly, leading todestruction of pancreatic islets and diabetes starting at 12-15 weeks ofage. B cell depletion using anti-CD20 antibody in the early stage ofinsulitis has been reported to delay the disease onset (Hu et al., JClin Inves. 117, 3857 (2007)), suggesting that B cells play a criticalrole in the disease pathogenesis in NOD mice.

To test the activity of anti-muCD37 antibody, the 252-3 antibody wasinjected into six female NOD mice intraperitoneally at 25 mg/kg every 10days for a total of 4 injections starting at 5 weeks of age (n=6). Thecontrol mice (n=6) were injected with polyclonal murine IgG (JacksonImmunoresearch, West Grove, Pa.). Three days after the last injection,the B and T cell levels in peripheral blood were examined by flowcytometry. Allophycocyanin (APC)-conjugated anti-mouse CD45R (B220)antibody (ebioscience, San Diego, Calif.) and fluorescein isothiocyanate(FITC)-conjugated anti CD3c antibody (ebioscience, San Diego, Calif.)were used to stain B and T cell populations, respectively. The B/T cellratio was normalized to murine IgG control treated samples as describedabove and the normalized B/T cell ratio was plotted for muIgG controlmice and 252-3 antibody treated mice (FIG. 10A). The results show thatthe B cell level of the mice treated with 252-3 antibody wassignificantly reduced as compared to the control mice, suggesting thatthe 252-3 antibody efficiently depletes peripheral blood B cells in NODmice. To examine the effect of B cell depletion by anti-muCD37 antibody,blood glucose level was measured weekly starting at 12 weeks of age.Mice with blood glucose level ≥250 mg/dL in two consecutive weeks areconsidered diabetic. The data in FIG. 10B shows that the control micestarted to develop diabetes on week 15 and 83% of the mice had diabeteson week 22. In contrast, the mice treated with 252-3 antibody started todevelop diabetes on week 17 and only 50% of the mice were diabetic onweek 27. This data shows that treatment of 252-3 antibody efficientlydepletes B cells in NOD mice, delays the onset of diabetes andsignificantly reduces the disease incidence.

Example 7 Anti-muCD37 Monoclonal Antibody Inhibits Collagen-InducedArthritis

Collagen-induced arthritis (CIA) is an animal model of rheumatoidarthritis (RA) that is widely used to investigate disease pathogenesisand to validate therapeutic targets. Arthritis is normally induced inmice or rats by immunization with autologous or heterologous type IIcollagen in adjuvant. This immunization elicits a robust T- and B-cellresponse to the antigen leading to proliferative synovitis withinfiltration of polymorphonuclear and mononuclear cells, pannusformation, cartilage degradation, bone erosion and fibrosis.

Since different mouse strains have different susceptibility toantibody-mediated B cell depletion (Ahuja et al., J. Immunol., 179:3351-3361 (2007)), to test the activity of anti-muCD37 antibody in CIAmodel, we first studied the capacity of the 252-3 antibody to deplete Bcells in DBA/1 mice. Mice were injected intraperitoneally with 25 mg/kgof 252-3 antibody or polyclonal murine IgG (Jackson Immunoresearch, WestGrove, Pa.) as control. Peripheral blood was collected at different timepoints and analyzed for B and T cell levels by flow cytometry.Allophycocyanin (APC)-conjugated anti-mouse CD45R (B220) antibody(ebioscience, San Diego, Calif.) and fluorescein isothiocyanate(FITC)-conjugated anti CD3c antibody (ebioscience, San Diego, Calif.)were used to stain B and T cell populations, respectively. Thenormalized B/T cell ratio was calculated as described above and comparedbetween the muIgG control mice and 252-3 antibody treated mice (FIG.11A). The result show that the 252-3 antibody significantly reduced theperipheral blood B cell level to ˜20% and ˜8% in 1 and 3 days after theantibody injection, and this low B cell level was maintained at 7 daysafter the antibody injection. This data suggests that the 252-3 antibodycan rapidly and efficiently deplete peripheral blood B cells, and thiseffect was sustained for at least 7 days after the antibody injection.

The second study tests the capacity of 252-3 antibody to inhibit CIA. Inthis study, CIA was induced in DBA/1 mice by subcutaneous immunizationof chicken collagen/CFA (complete Freund's adjuvant) on day 0 andchicken collagen/IFA (incomplete Freund's adjuvant) on day 21 (HookeLaboratories, Lawrence, Mass.). Mice were checked for CIA signs dailystarting on day 21 after immunization. The CIA severity was scored on ascale of 0 to 16 (based on a score of 0 to 4 for each paw) using thefollowing criteria:

Paw Score Clinical Observations 0 Normal paw. 1 One toe inflamed andswollen. 2 More than one toe, but not entire paw, inflamed and swollen,OR Mild swelling of entire paw. 3 Entire paw inflamed and swollen. 4Very inflamed and swollen paw or ankylosed paw. If the paw is ankylosed,the mouse cannot grip the wire top of the cage.

At the onset of arthritis symptoms, mice were randomized into two groupsand injected with the 252-3 antibody or polyclonal muIgGintraperitoneally at 10 mg/kg dose at three consecutive days. A total of12 mice were enrolled for each group. At the end of the study (21 daysafter the disease onset), the data were synchronized based on the day ofdisease onset for each mouse. The disease progression plot (FIG. 11B)shows that the disease severity in control mice increased rapidly frommean score of 2 at day 1 to 9.5 at day 7. In contrast, the disease inmice treated with the 252-3 antibody progressed significantly slowerwith mean score of 4.4 at day 7. Altogether, this data suggests that the252-2 antibody treatment significantly depletes the B cell populationand alleviates CIA.

In conclusion, the above experiments using a surrogate anti-muCD37antibody provide evidence that a CD37-targeting antibody, or animmunoconjugate that includes a CD37 antibody, can inhibit autoimmunediseases in animal models.

It is to be appreciated that the Detailed Description section, and notthe Abstract section, is intended to be used to interpret the claims.The Abstract may set forth one or more but not all exemplary embodimentsof the present invention as contemplated by the inventors, and thus, isnot intended to limit the present invention and the appended claims inany way.

The present invention has been described above with the aid offunctional building blocks illustrating the implementation of specifiedfunctions and relationships thereof. The boundaries of these functionalbuilding blocks have been arbitrarily defined herein for the convenienceof the description. Alternate boundaries can be defined so long as thespecified functions and relationships thereof are appropriatelyperformed.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications such specific embodiments, without undueexperimentation, without departing from the general concept of thepresent invention. Therefore, such adaptations and modifications areintended to be within the meaning and range of equivalents of thedisclosed embodiments, based on the teaching and guidance presentedherein. It is to be understood that the phraseology or terminologyherein is for the purpose of description and not of limitation, suchthat the terminology or phraseology of the present specification is tobe interpreted by the skilled artisan in light of the teachings andguidance.

The breadth and scope of the present invention should not be limited byany of the above-described exemplary embodiments, but should be definedonly in accordance with the following claims and their equivalents.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each independent publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

What is claimed is:
 1. An antibody or antigen binding fragment thereofthat specifically binds to CD37, wherein said antibody or fragmentcomprises a variable heavy chain CDR1 comprising the amino acid sequenceof SEQ ID NO:171, a variable heavy chain CDR2 comprising the amino acidsequence of SEQ ID NO:172 or SEQ ID NO:181, and a variable heavy chainCDR3 comprising the amino acid sequence of SEQ ID NO:173, a variablelight chain CDR1 comprising the amino acid sequence of SEQ ID NO:174, avariable light chain CDR2 comprising the amino acid sequence of SEQ IDNO:175, and a variable light chain CDR3 comprising the amino acidsequence of SEQ ID NO:176.
 2. The antibody or antigen-binding fragmentthereof of claim 1, wherein said antibody or antigen binding fragmentthereof is murine, non-human, humanized, chimeric, resurfaced, or human.3. The antibody or antigen-binding fragment thereof of claim 1, which isa full length antibody.
 4. The antibody or antigen-binding fragmentthereof of claim 1, which is an antigen-binding fragment.
 5. Theantibody or antigen-binding fragment thereof of claim 1, wherein saidantibody or antigen-binding fragment thereof comprises a Fab, Fab′,F(ab′)2, single chain Fv or scFv, disulfide linked Fv, intrabody,IgGΔCH2, minibody, F(ab′)3, tetrabody, triabody, diabody, DVD-Ig, mAb2,(scFv)2, or scFv-Fc.
 6. An immunoconjugate comprising the antibody orantigen binding fragment of claim 1, a linker, and a cytotoxic agent. 7.The immunoconjugate of claim 6, wherein said linker is selected from thegroup consisting of a cleavable linker, a non-cleavable linker, ahydrophilic linker, and a dicarboxylic acid based linker.
 8. Theimmunoconjugate of claim 6, wherein said linker is selected from thegroup consisting of: N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP);N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB) or N-succinimidyl4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB); N-succinimidyl4-(maleimidomethyl) cyclohexanecarboxylate (SMCC); N-sulfosuccinimidyl4-(maleimidomethyl) cyclohexanecarboxylate (sulfoSMCC);N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB); andN-succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol] ester(NHS-PEG4-maleimide).
 9. The immunoconjugate of claim 6, wherein saidcytotoxic agent is selected from the group consisting of a maytansinoid,maytansinoid analog, doxorubicin, a modified doxorubicin,benzodiazepine, taxoid, CC-1065, CC-1065 analog, duocarmycin,duocarmycin analog, calicheamicin, dolastatin, dolastatin analog,auristatin, tomaymycin derivative, and leptomycin derivative or aprodrug of the agent.
 10. The immunoconjugate of claim 9, wherein saidcytotoxic agent is a maytansinoid.
 11. The immunoconjugate of claim 9,wherein said cytotoxic agent isN(2′)-deacetyl-N(2′)-(3-mercapto-1-oxopropyl)-maytansine (DM1) orN(2′)-deacetyl-N(2′)-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4).12. The immunoconjugate of claim 6, wherein the immunoconjugatecomprises 3-4 cytotoxic agents.
 13. The antibody or antigen-bindingfragment of claim 1, wherein said antibody or fragment comprises thepolypeptide sequences of SEQ ID NO:177 and SEQ ID NO:178.
 14. Animmunoconjugate comprising the antibody or antigen binding fragment ofclaim 13, a linker, and a cytotoxic agent.
 15. The immunoconjugate ofclaim 14, wherein the cytotoxic agent is a maytansinoid.
 16. Theimmunoconjugate of claim 15, wherein the maytansinoid isN(2′)-deacetyl-N(2′)-(3-mercapto-1-oxopropyl)-maytansine (DM1) orN(2′)-deacetyl-N(2′)-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4).17. The immunoconjugate of claim 14, wherein the immunoconjugatecomprises 3-4 cytotoxic agents.
 18. The antibody or antigen-bindingfragment of claim 1, wherein said antibody or fragment comprises thepolypeptide sequences of SEQ ID NO:179 and SEQ ID NO:180.
 19. A methodfor depleting a B-cell comprising contacting a population of cellscomprising a B-cell with the antibody or antigen-binding fragment ofclaim 1, wherein the contacting results in depletion of the B-cell. 20.A method for depleting a B-cell comprising contacting a population ofcells comprising a B-cell with the immunoconjugate of claim 6, whereinthe contacting results in depletion of the B-cell.